Compounds for inducing proliferation and differentiation of cells, and methods of use thereof

ABSTRACT

The present invention provides methods of inducing proliferation of and/or differentiating cells comprising contacting cells with compounds within the methods of the invention. The present invention further provides cells obtainable by the methods of the invention. Liver disease affects more than 500 million people worldwide. Organ transplantation is the gold standard for treatment of liver failure, but organ shortages are acute.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the U.S. national phase application, pursuantto 35 U.S.C. § 371, of PCT international application Ser. No.PCT/US2014/028408, filed Mar. 14, 2014, designating the United Statesand published in English, which claims priority under 35 U.S.C. § 119(e)to U.S. Provisional Application No. 61/798,902, filed Mar. 15, 2013,which applications are hereby incorporated by reference in their itsentirety herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under grant numbersHG005032, DK065152, and DK56966 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Liver disease affects more than 500 million people worldwide. Organtransplantation is the gold standard for treatment of liver failure, butorgan shortages are acute. Cell-based therapies, such as celltransplantation, engineered hepatocellular tissue constructs andbioartificial liver devices, have long held promise as alternatives towhole organ transplantation. Such therapies require the use of humanhepatocytes due to substantial species-specific differences betweenanimal and human hepatocellular functions.

Human hepatocytes, within the stromal context of the liver in vivo, arecapable of extensive proliferation, enough to fully regenerate the liverfollowing up to 66% partial hepatectomy. However, this ability is lostin vitro, despite extensive gene-by-gene and protein-by-proteinapproaches towards harnessing this tremendous replication potential ofhuman hepatocytes. Investigations have yielded many different cultureconditions that can support moderate expansion of mouse and rathepatocytes, including a multi-factor media formulation that expands rathepatocytes through a dedifferentiated bi-potential intermediate.However, translation of these findings to human cultures has not beenreported. Human cells are critical for cell-based therapies due tosubstantial differences between animal and human hepatocellularfunctions, including apolipoprotein expression, metabolic regulation ofcholesterol, and phase I detoxification enzymes. To overcome the growthlimitations of primary human cells, various human hepatocyte cell lineshave been developed. Although these cell lines are growth-competent,they introduce safety concerns and underperform primary cells in termsof liver function.

In addition to their use in the transplantation therapies to treat liverdiseases, human hepatocytes are in high demand for drug toxicityscreening and development, because of their critical function in thedetoxification of drugs or other xenobiotics, as well as endogenoussubstrates. However, human primary hepatocytes quickly lose theirfunctions when cultured in vitro. Moreover, the drug metabolic abilityof human primary hepatocytes exhibits significant variance amongindividuals. The availability of an unlimited supply of patient-specificfunctional hepatocytes would greatly facilitate both the drugdevelopment and the eventual clinical application of hepatocytetransplantation.

Alternative sources for hepatic cells are being investigated, such asvarious stem cell populations. Stem cells hold great promise as abiologics source due to their ability to self-renew without limit and todifferentiate along many lineages. Induced pluripotent stem cells (iPScells) additionally create the possibility of establishingpatient-specific cell types, thus empowering personalized medicine.Human iPS cells are generated from somatic cells via forced expressionof reprogramming factors, and can be differentiated towardshepatocyte-like cells (iHeps) in a step-wise manner, using definedfactors. Mouse iHeps can also be generated directly from fibroblasts viacellular reprogramming. Although diverse stem and progenitor cell typesexhibit vast potential for integration into hepatic treatments, manychallenges remain, including the ability to completely dictatedifferentiation into fully mature hepatocytes. While human iHeps closelyresemble mature hepatocytes, key differences in their phenotypes tlimittheir use as a renewable source of functional hepatocytes. Notably,iHeps persistently express fetal markers like alpha fetoprotein (AFP)and lack key mature hepatocyte functions, as reflected by drasticallyreduced activity (0.1%) of many CYP450s (e.g. CYP2A6 and CYP3A4).Consequently, for decades, human hepatocyte sourcing has been abottleneck for many fields of research and clinical therapies.

There is a need for identifying compounds that induce proliferationand/or differentiation of cells, such as somatic cells. Such compoundsmay be used, for example, to produce hepatic lineage cells fortherapeutic and research use, such as human hepatocytes. The presentinvention addresses and satisfies this unmet need.

BRIEF SUMMARY OF THE INVENTION

As described herein, the present invention provides compounds thatinduce proliferation of cells, such as hepatocytes, in vitro or in vivo,and/or induces differentiation of pluripotent stem cells so as toprovide functional cells, such as functional hepatocytes. The presentinvention further provides methods of inducing proliferation ofhepatocytes in vitro or in vivo, and/or inducing differentiation ofpluripotent stem cells, and cells prepared according to the methods ofthe invention.

The invention provides a method of inducing proliferation of one or moreprimary cells, the method comprising contacting the one or more primarycells with a compound of the invention.

The invention further provides a method of inducing differentiation ofone or more induced pluripotent stem cells (iPS cell), the methodcomprising contacting the one or more iPS cells with at least onecompound of the invention, whereby contacting the one or more iPS cellswith the at least one compound induces differentiation of the one ormore iPS cells.

The invention further provides a cell produced by a method of theinvention.

The invention further provides a method of treating, alleviating orpreventing a disease or condition in a subject in need thereof, themethod comprising administering to a subject a therapeutically effectiveamount of at least one agent selected from the group consisting of: (i)at least one compound of the invention; (ii) one or more cells obtainedby a method of the invention; (iii) one or more cells of the invention;and, any combinations thereof.

The invention further provides a method of performing tissue or organtransplant in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of atleast one agent selected from the group consisting of: (i) at least onecompound of the invention; (ii) one or more cells obtained by a methodof the invention; (iii) one or more cells of the invention; and, anycombinations thereof.

The invention further provides a method of repairing damaged, diseasedor aged tissue in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of atleast one agent selected from the group consisting of: (i) at least onecompound of the invention; (ii) one or more cells obtained by a methodof the invention; (iii) one or more cells of the invention; and, anycombinations thereof.

The invention further provides a method of providing cell replacementtherapy to a subject in need thereof, the method comprising: (i)contacting one or more cells with at least one compound of theinvention, thereby generating one or more differentiated cells; and (ii)administering the one or more differentiated cells to the subject.

The invention further provides a method of developing a humanized mousemodel, the method comprising administering to a mouse at least one cellselected from the group consisting of (i) one or more cells obtained bya method of the invention; (ii) one or more cells of the invention; andany combinations thereof.

In various embodiments of any of the above aspects or any other aspectof the invention delineated herein, the compound is selected from thegroup consisting of:

-   Compound 109 (PH1, or    4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one);-   Compound 110    ((1R,4R)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexane-1-carboxamide);-   Compound 111    ((1R,4R)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexane    carboxamide);-   Compound 112    (1-((9H-carbazol-4-yl)oxy)-3-((2-(2-methoxyphenoxy)ethyl)amino)    propan-2-ol);-   Compound 113    (3-(5H-dibenzo[a,d][7]annulen-5-ylidene)-N,N-dimethylpropan-1-amine);-   Compound 114 (2-(2-(1,3-dioxoisoindolin-2-yl)acetamido)acetic acid);    a compound of Formula (I):

wherein in (I):

-   -   W is H, F, Cl, Br, I, OH, lower alkyl or lower alkoxy; X is F,        Cl, Br or I; Y and Z are independently C(═O) or S(═O)₂; R¹ is        lower alkyl; each occurrence of R² is independently selected        from the group consisting of H, OH, F, Cl, Br, I, alkyl,        perfluoroalkyl, alkoxy, perfluoroalkoxy, NH₂, acylamino, amido,        carboxyl, alkoxycarbonyl, acyloxy, formyl, acyl, thioester,        carbamate, urea, sulfonate, sulfamoyl, sulfone, sulfonamide, CN,        NO₂, and alkylthio; and, n is 0, 1, 2, 3, 4 or 5;        a compound of Formula (II):

wherein in (II):

-   -   W is H, F, Cl, Br, I, OH, lower alkyl or lower alkoxy; X is F,        Cl, Br or I; each occurrence of R is independently H or lower        alkyl; R³¹ is H or lower alkyl; and, R³² is amido, carboxyl,        alkoxycarbonyl or sulfonamide;        a compound of Formula (III):

wherein in (III):

-   -   G is C(R)₂, O or N(R); each occurrence of Q is independently O        or N(R); each occurrence of R is independently H or lower alkyl;        each occurrence of Y is independently C(═O) or S(O)₂; and, R⁴¹        is lower alkyl or lower alkoxy;        a compound of Formula (IV):

wherein in (IV):

-   -   each occurrence of R is independently H or lower alkyl; Q is a        bond, O or N(R); Y is C(═O) or S(O)₂; R¹¹ is substituted or        unsubstituted alkyl, cycloalkyl, aryl, heteroaryl or aralkyl;        R¹² is H or lower alkyl; R¹³ is H or substituted or        unsubstituted lower alkyl; R¹⁴ is H, F, Cl, Br, I, substituted        or unsubstituted aryl or heteroaryl, or —C≡C—R¹⁶, where R¹⁶ is        substituted or unsubstituted aminoalkyl, alkoxyalkyl, aryl or        heteroaryl; each occurrence of R¹⁵ is independently OH, F, Cl.        Br, I, NH₂, CN, NO₂, lower alkyl, or lower alkoxy; and, n is 0,        1 or 2;        a compound of Formula (V):

wherein in (V):

-   -   each occurrence of R is independently H or lower alkyl; Q is a        bond, O or NR; Y is C═O or S(O)₂, preferably C═O; R²¹ is        substituted or unsubstituted alkyl, aralkyl, cycloalkyl, aryl or        heteroaryl; R²² is H or lower alkyl; R²³ is H or substituted or        unsubstituted lower alkyl; and, R²⁵ is H, OH, F, Cl, Br, I,        lower alkyl, lower alkoxy, NH₂, CN, or NO₂;        enantiomers or diastereoisomers thereof, prodrugs thereof,        pharmaceutically acceptable salts thereof, and any combinations        thereof.

In certain embodiments, the at least one compound is selected from thegroup consisting of Compounds 109-114, a compound of Formula (I)-(V),enantiomers or diastereoisomers thereof, prodrugs thereof,pharmaceutically acceptable salts thereof and any combinations thereof.

In certain embodiments, the at least one compound is selected from thegroup consisting of:

compound nomenclature 1012-(N-(5-chloro-2-methylphenyl)methylsulfonamido)-N-(2,6-difluorophenyl)acetamide(FPH2) 102 2-(N-(5-chloro-2-methylphenyl)methylsulfonamido)-N-(2-(methylthio)phenyl)acetamide 103N-(4-bromo-3-methylphenyl)-2-(N-(5-chloro-2-methylphenyl)methylsulfonamido)acetamide 1044-(3-(5-chloro-2-methoxyphenyl)thioureido)-1-ethyl-1H-pyrazole-3-carboxamide(FPH1) 105 N,N′-(methylenebis(4,1-phenylene))diacetamide (FH1) 1061-(((2R,3S)-8-(benzofuran-2-yl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 1073-(4-fluorophenyl)-1-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 108N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide109 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (PH1) 110(1R,4R)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexane-1-carboxamide111 (1R,4R)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide1121-((9H-carbazol-4-yl)oxy)-3-((2-(2-methoxyphenoxy)ethyl)amino)propan-2-ol113 3-(5H-dibenzo[a,d][7]annulen-5-ylidene)-N,N-dimethylpropan-1-amine114 2-(2-(1,3-dioxoisoindolin-2-yl)acetamido)acetic acid 2011-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-isopropyl-1-methylurea 2021-(((2R,3S)-8-((3,4-dimethoxyphenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 2032-fluoro-N-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-N-methylbenzamide 2041-(((2R,3S)-8-(3-cyclopentylprop-1-yn-1-yl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 2053-(4-fluorophenyl)-1-(((2R,3R)-8-((4-fluorophenyl)ethynyl)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 206N-(((4R,5R,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide2073-(4-fluorophenyl)-1-(((2R,3S)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-8-phenyl-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea208isobutyl(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)(methyl)carbamate 2091-(((2R,3S)-8-(4-cyanophenyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 210N-(((4S,5R,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide211N-(((4R,5S,Z)-7-((R)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide212N-(((4S,5R,Z)-7-((R)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide215N-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-2-methoxy-N-methylacetamide 217isopropyl(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)(methyl)carbamate 2243-(4-fluorophenyl)-1-(((2R,3S)-8-((3-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 2254-(dimethylamino)-N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylbutanamide226N-(((4S,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide227N-(((4S,5S,Z)-7-((R)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide228N-(((4R,5R,Z)-7-((R)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide2291-(((2R,3S)-8-(cyclopropylethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 2303-(4-fluorophenyl)-1-(((2R,3S)-8-((R)-3-hydroxybut-1-yn-1-yl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 2311-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methyl-3-phenylurea 2323-(4-fluorophenyl)-1-(((2S,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 2333-(4-fluorophenyl)-1-(((2R,3R)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 2343-(4-fluorophenyl)-1-(((2S,3R)-8-((4-fluorophenyl)ethynyl)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 2353-(4-fluorophenyl)-1-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 2363-(4-fluorophenyl)-1-(((2R,3S)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 2371-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-methoxyphenyl)-1-methylurea 2383-(4-fluorophenyl)-1-(((2S,3S)-8-((4-fluorophenyl)ethynyl)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 2393-(4-fluorophenyl)-1-(((2S,3R)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 240N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclohexanecarboxamide 241N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methyl-2-phenylacetamide 242N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylbenzamide 2431-(((2R,3S)-8-(3-(dimethylamino)prop-1-yn-1-yl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 2441-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methyl-3-(naphthalen-1-yl)urea 245N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N,1-dimethyl-1H-indole-6-carboxamide246N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methyl-5-phenylisoxazole-3-carboxamide247N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylisonicotinamide 2483-(3,5-dimethylisoxazol-4-yl)-1-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 2493-(4-fluorophenyl)-1-(((2R,3S)-8-(3-hydroxyhex-1-yn-1-yl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 250benzyl(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)(methyl)carbamate 251ethyl(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)(methyl)carbamate 2523-(4-fluorophenyl)-1-(((2R,3S)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-8-(phenylethynyl)-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 2531-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-3-isopropyl-1-methylurea 2543-(4-fluorophenyl)-1-(((2R,3S)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-8-((4-(trifluoromethyl)phenyl)ethynyl)-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 2551-(((2R,3S)-8-bromo-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 2564,4,4-trifluoro-N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylbutanamide2573-(4-fluorophenyl)-1-(((2R,3S)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-8-(3-(trifluoromethyl)phenyl)-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylureaenantiomers or diastereoisomers thereof, prodrugs thereof,pharmaceutically acceptable salts thereof, and any combinations thereof.

In certain embodiments, the at least one compound is selected from thegroup consisting of Compounds 101-114, 201-212, 215, 217, 224-257,enantiomers or diastereoisomers thereof, prodrugs thereof,pharmaceutically acceptable salts thereof and any combinations thereof.

In certain embodiments, the one or more primary cells comprise one ormore somatic cells. In other embodiments, the one or more primary cellsare obtained from a subject or are part of a subject's body. In yetother embodiments, the one or more primary cells comprise one or moreadult cells. In yet other embodiments, the one or more primary cellscomprise one or more epithelial cells or endothelial cells. In yet otherembodiments, the one or more primary cells comprise one or morehepatocytes.

In certain embodiments, the one or more primary cells comprise one ormore stem cells. In other embodiments, the one or more stem cellscomprise a pluripotent or non-pluripotent cell. In yet otherembodiments, the one or more stem cells are selected from the groupconsisting of a multipotent stem cell, oligopotent stem cell, unipotentstem cell, and any combinations thereof. In yet other embodiments, thepluripotent stem cell is selected from the group consisting of inducedpluripotent stem cell; embryonic stem cell; pluripotent stem cellderived by nuclear transfer, cell fusion, or forced expression ofreprogramming factors; and any combinations thereof. In yet otherembodiments, the one or more stem cells comprise a fetal stem cell oradult stem cell. In yet other embodiments, the one or more stem cellscomprise an epithelial stem cell or endothelial stem cell.

In certain embodiments, the one or more iPS cells are differentiatedinto one or more hepatocyte-like cells (iHep cells). In otherembodiments, the one or more iHep cells are contacted on day 21 to day35 after the differentiation with at least one compound of theinvention. In yet other embodiments, the one or more iHep cells arecontacted with at least one compound of the invention after thedifferentiation for a period of at least 5 days.

In various embodiments of any of the above aspects or any other aspectof the invention delineated herein, the compounds of the inventioninduce functional proliferation of endogenous cells. In otherembodiments, the compounds of the invention promote differentiation ofendogenous cells. In yet other embodiments, the disease or conditioncomprises liver disease. In yet other embodiments, the liver disease isselected from acetaminophen toxicity, alcoholic liver disease, primaryliver cancer, liver cirrhosis, liver cysts, fatty liver disease, liverfibrosis, hepatitis, primary sclerosing cholangitis, jaundice, and anycombinations thereof. In yet other embodiments, the organ comprisesliver. In yet other embodiments, the tissue comprises liver tissue. Inyet other embodiments, one or more cells are obtained from the subject.

In various embodiments of any of the above aspects or any other aspectof the invention delineated herein, a bio-artificial liver devicecomprising one or more cells of the invention is implanted in thesubject. In other embodiments, the subject is further administered aneffective amount of at least one compound of the invention.

In certain embodiments, the hepatitis is caused by a virus selected fromthe group consisting of hepatitis A-E virus, herpes simplex,cytomegalovirus, Epstein-Barr virus, yellow fever, and any combinationsthereof. In other embodiments, the at least one compound is administeredby a route selected from the group consisting of oral, parenteral,intramuscular, intranasal, sublingual, intratracheal, inhalation,ocular, vaginal, rectal, and intracerebroventricular, and anycombinations thereof. In yet other embodiments, the subject is a human.

Other aspects, embodiments, advantages, and features of the presentinvention will become apparent from the following specification.

BRIEF DESCRIPTION OF THE FIGURES

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention,specific embodiments are illustrated in the drawings. It should beunderstood, however, that the invention is not limited to the precisearrangements and instrumentalities of the embodiments shown in thedrawings.

FIG. 1, comprising FIGS. 1A-1B, is a set of images and graphsillustrating a representative screening of the invention. FIG. 1A topcomprises an image illustrating the design of the screening platform,which includes a sparse population of hepatocytes co-cultivated on topof a confluent layer of J2-3T3 fibroblasts within 384-well plates. FIG.1A bottom comprises a series of graphs and high content imagesillustrating the finding that compounds of the invention stabilizedhepatocyte phenotypic function in vitro (Example 2). FIG. 1B topcomprises an illustration of the workflow of the primary screening ofsmall molecules. FIG. 1B bottom comprises a set of bar graphsillustrating the type of compounds that constituted the initial set of93 compounds that met all hit selection criteria qualifying asfunctional proliferation hits (FPHs) and also scatterplots of primaryscreening data (Example 3).

FIG. 2, comprising FIGS. 2A-2H, illustrates the results of treatment ofhepatocytes with FPHs. FIG. 2A comprises a bar graph (top) and images(bottom) illustrating that FPH2 induced a 1.5 fold increase inhepatocyte nuclei numbers during the primary screening (Example 4). FIG.2B comprises a bar graph (left) and images (right) illustrating thattreatment of hepatocyte with FPH2 elevated the number of hepatocytenuclei in culture and the number of nuclei undergoing mitosis (Example4). FIG. 2C comprises a dose-response graph illustrating that theeffects of FPH2 on hepatocytes were dose-responsive (Example 4). FIG. 2Dcomprises a series of phase contrast images illustrating that hepatocytemorphology remained normal throughout the treatment period (Example 4).FIG. 2E comprises a series of images illustrating the effects of FPH1and FPH2 on human primary hepatocytes cultured for seven days andtreated with compound on days 1 and 5 at a concentration of 20 μM(Example 4). FIG. 2F comprises a set of bar graphs showing an increasein the area of albumin-positive colonies in FPH1- and FPH2-treatedcultures (Example 4). FIG. 2G comprises a set of bar graphs and FIG. 2Hcomprises a set of FACS plots illustrating that there was up to 10 foldincrease in the number of hepatocytes when treated with FPH compounds(Example 4).

FIG. 3, comprising FIGS. 3A-3C, illustrates the characterization ofhepatocytes treated with compounds of the invention. FIG. 3A comprises aseries of bar graphs illustrating that albumin secretion, urea synthesisand CYP450 activity were all normal throughout FPH treatment (Example4). FIG. 3B comprises a set of images illustrating active transport offluorometric substrate into the bile cannaliculi between hepatocytes(Example 4). FIG. 3C comprises graphs and summaries of gene expressionprofiles illustrating that the profiles of treated hepatocytes moreclosely resembled mature hepatocytes than untreated controls (Example4).

FIG. 4, comprising FIGS. 4A-4F, illustrates characterization ofhepatocytes treated with compounds of the invention. FIG. 4A (left)comprises a bar graph illustrating that FH1 doubled albumin secretedduring primary screening and FIG. 4A (right) comprises a graphillustrating that the effects were dose responsive (Example 5). FIG. 4Bis a set of images illustrating that colonies of hepatocyte-like cellsincreased in size and improved in morphology with treatment of compoundsof the invention (Example 5). FIG. 4C comprises a series of graphs andgene expression profiles illustrating that the profiles of treated iHepsmore closely resembled mature hepatocytes than untreated (Example 5).FIG. 4D comprises a series of images (top) and bar graphs (bottom)illustrating dramatic increases of albumin and CYP3A staining upon FH1and FPH2 treatment. There was also marked decrease of AFP staining(Example 5). FIG. 4E comprises a set of graphs illustrating that AFPsecretion decreased while albumin secretion, CYP3A4 and CYP2A6 activityincreased upon treatment with a compound of the invention (Example 5).FIG. 4F comprises a a set of graphs relating to hepatocyte inductionkinetics with black arrows indicating addition of the inducer and whitearrows indicating removal of the inducer (Example 5).

FIG. 5, comprising FIGS. 5A-5C, illustrates the effects of treatinginduced hepatocyte-like cells (iHeps) with compounds of the invention.FIG. 5A comprises a series of images illustrating that treated iHepsmaintained mature phenotypes for 9 days after removal of FPH2 and FH1(Example 5). FIG. 5B comprises a set of bar graphs illustrating thattreated iHeps maintained elevated expression of the mature phenotypicmarker albumin and minimal expression of the fetal markers AFP for 9days after removal of FPH2 and FH1 (Example 5). FIG. 5C comprises a setof bar graphs illustrating that treated iHeps maintained elevated levelsof CYP34A and CYP2A6 activity for 9 days after removal of FPH2 and FH1(Example 5).

FIG. 6, comprising FIGS. 6A-6B, illustrates the effects of compounds ofthe invention on primary human hepatocytes. FIG. 6A comprises a seriesof images illustrating the effects of FPH1 and FPH2 on multipledifferent donors of primary human hepatocytes (Example 6). FIG. 6Bcomprises a set of bar graphs illustrating the quantification ofhepatocytes in FPH treated and control cultures (Example 6).

FIG. 7, comprising FIGS. 7A-7C, illustrates iPS and iHep cells. FIG. 7Acomprises a set of phase contrast images of iPS and iHep cells inculture (Example 7). FIG. 7B comprises a series of images illustratingthe expression of hepatic lineage markers in iPS and iHep cells inculture (Example 7). FIG. 7C comprises a series of FACS plotsillustrating the expression profile of hepatic and immature lineagemarkers in iPS and iHep cells in culture (Example 7).

FIG. 8, comprising FIGS. 8A-8D, illustrates experiments in whichzebrafish were treated with compounds of the invention. FIG. 8Acomprises an image illustrating the experimental protocol of compoundtreatment in zebrafish (Example 8). FIGS. 8B-8C comprise imagesillustrating that treated zebrafish had larger livers compared tocontrols (Example 8). FIG. 8D comprises a bar graph showing the increasein liver size of treated zebrafish compared to controls (Example 8).

FIG. 9, comprising FIGS. 9A-9D, illustrates experiments in whichzebrafish were treated with toxic amounts of acetyl-para-aminophenol(APAP). FIG. 9A comprises an image illustrating the experimentalprotocol for APAP induced toxicity-induced in zebrafish embryos (Example9). FIG. 9B comprises a set of graphs illustrating the percentage ofzebrafish embryos that survived a fatal dose of APAP (Example 9). FIG.9C comprises an image illustrating the experimental protocol for APAPinduced toxicity-induced in adult zebrafish (Example 9). FIG. 9Dcomprises a graph illustrating the percentage of adult zebrafish thatsurvived a fatal dose of APAP (Example 9).

FIG. 10, comprising FIGS. 10A-10B, illustrates experiments in whichzebrafish were treated with acetyl-para-aminophenol (APAP). FIG. 10Acomprises an image illustrating the experimental protocol for APAPinduced toxicity-induced in adult zebrafish and assessment oftherapeutic window (Example 9). FIG. 10B comprises a series of imagesillustrating that FH1 and PH1 enhance embryonic liver size following anon-fatal dose of APAP (Example 9).

FIG. 11, comprising FIGS. 11A-11F, illustrates the activity of compoundsof the invention on hepatocytes. FIG. 11A comprises a bar graphillustrating that compounds 106, 107 and 108 induced proliferation ofhepatocytes (Example 10). FIG. 11B comprises a series of images ofcannaliculi staining of compounds 106-, 107- and 108-treated hepatocytesindicated that cells exhibit functional liver phenotypes (Example 10).FIG. 11C comprises a bar graph showing treated hepatocytes haveincreased CYP450 activity (Example 10). FIG. 11D comprises a set ofgraphs and summary of gene expression profiles illustrating that theprofiles of treated hepatocytes more closely resembled maturehepatocytes than untreated controls (Example 10). FIG. 11E comprises aseries of images illustrating that compound 107 treatment increased Ki67staining, which not only co-localized with Hoechst stains for cellnuclei but also with human albumin stains for hepatocytes. FIG. 11Fcomprises a set of tables illustrating the effects of compounds onhepatocyte proliferation (Example 10).

FIG. 12, comprising FIGS. 12A-12C, illustrates the effects of compoundsof the invention on iHeps. FIG. 12A comprises a set of graphs, FIG. 12Bcomprises a set of images, and FIG. 12C comprises a set of tablesillustrating that treated iHeps develop more mature hepatocytephenotypes (Example 11).

FIG. 13 comprises a set of images illustrating effects of compounds ofthe invention on differentiation of iPS-derived endothelial cells(Example 12).

FIG. 14, comprising FIGS. 14A-14C, comprises a set of plots illustratinganalytical data for FPH1: liquid chromatography (FIG. 14A) and NMR(FIGS. 14B-14C).

FIG. 15, comprising FIGS. 15A-15C, comprises a set of plots illustratinganalytical data for FPH2: liquid chromatography (FIG. 15A) and NMR(FIGS. 15B-15C).

FIG. 16 comprises a plot illustrating liquid chromatography data forFH1.

FIG. 17 comprises a set of plots illustrating the kinome analyses forselected compounds of the invention.

FIG. 18 is a bar graph illustrating the in vivo therapeutic effects ofselected compounds of the invention in a zebrafish model ofacetaminophen overdose. Compounds tested were DOS 0 (BRD-K05085281;106), DOS 1 (BRD-K17976466; 108), and DOS 3 (BRD-K37628956; 107).

FIG. 19, comprising FIGS. 19A-19B, is a set of bar graphs illustratingthe in vitro maturation effects of selected compounds of the inventionon human iPS-derived hepatocyte-like cells (iHeps). FIG. 19A relates toa mature marker (albumin) and FIG. 19B relates to an immature marker(AFP). Compounds tested were DOS 1 (BRD-K17976466; 108), and DOS 3(BRD-K37628956; 107).

FIG. 20, comprising FIGS. 20A-20D, are a set of bar graphs illustratingthe cytochrome P450 activity in iHeps treated with compounds of theinvention. Compounds tested were DOS 1 (BRD-K17976466; 108), and DOS 3(BRD-K37628956; 107).

DETAILED DESCRIPTION OF THE INVENTION

Cell-based therapies hold the potential to alleviate the growing burdenof liver diseases. Such therapies require human hepatocytes, whosesourcing has limited medical and scientific research for decades. Thepresent invention overcomes several problems with current technologiesby providing methods and compounds for the growth and differentiation ofcells.

The present invention provides methods and compounds for the growth anddifferentiation of cells, such as hepatocytes. In certain aspects, thepresent invention relates to compounds that can be used to generatefunctional human hepatocytes. In other aspects, compounds of theinvention induce functional proliferation of hepatocytes in vitro or invivo, and can thus be used to expand mature human primary hepatocytes.In yet other aspects, compounds of the invention enhance the functionsof cultured hepatocytes, and thus can be used to differentiateiPS-derived hepatocytes toward a more differentiated/mature phenotype.In yet other aspects, compounds of the invention and the cells producedusing these compounds are useful for the treatment and prevention ofdisease.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

Unless specifically stated or obvious from context, as used herein, theterms “a,” “an” and “the” are understood to be singular or plural. Thus,for example, reference to “an amino acid substitution” includesreference to more than one amino acid substitution.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within two standard deviations of the mean. “About” canbe understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The term “acyl” refers to a group represented by the general formulahydrocarbyl-C(═O)—, preferably alkyl-C(═O)—.

The term “acylamino” refers to an amino group substituted with an acylgroup and may be represented, for example, by the formulahydrocarbyl-C(O)NH—.

The term “acyloxy” refers to a group represented by the general formulahydrocarbyl-C(═O)O—, preferably alkyl-C(═O)O—.

The term “alkoxy” refers to an alkyl group, preferably a lower alkylgroup, having an oxygen attached thereto. Representative alkoxy groupsinclude methoxy, ethoxy, propoxy, tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl-.

The term “alkenyl” as used herein refers to an aliphatic groupcontaining at least one double bond and is intended to include both“unsubstituted alkenyls” and “substituted alkenyls,” the latter of whichrefers to alkenyl moieties having substituents replacing a hydrogen onone or more carbons of the alkenyl group. Such substituents may occur onone or more carbons that are included or not included in one or moredouble bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed elsewhere herein, except where stabilityis prohibitive. For example, substitution of alkenyl groups by one ormore alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

An “alkyl” group or “alkane” is a straight chained or branchednon-aromatic hydrocarbon that is completely saturated. Typically, astraight chained or branched alkyl group has from 1 to about 20 carbonatoms, preferably from 1 to about 10 unless otherwise defined. Examplesof straight chained and branched alkyl groups include methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl,pentyl and octyl. A C₁-C₆ straight chained or branched alkyl group isalso referred to as a “lower alkyl” group.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls,” the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents, if nototherwise specified, can include, for example, a halogen, hydroxyl,carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl),thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido,amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, oraromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate. For instance, thesubstituents of a substituted alkyl may include substituted andunsubstituted forms of amino, azido, imino, amido, phosphoryl (includingphosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido,sulfamoyl and sulfonate), and silyl groups, as well as ethers,alkylthios, carbonyls (including ketones, aldehydes, carboxylates, andesters), —CF₃, —CN and the like. Exemplary substituted alkyls aredescribed elsewhere herein. Cycloalkyls can be further substituted withalkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substitutedalkyls, —CF₃, —CN, and the like.

The term “alkylamino” as used herein refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio” as used herein refers to a thiol group substitutedwith an alkyl group and may be represented by the general formulaalkyl-S—.

The term “alkynyl” as used herein refers to an aliphatic groupcontaining at least one triple bond and is intended to include both“unsubstituted alkynyls” and “substituted alkynyls,” the latter of whichrefers to alkynyl moieties having substituents replacing a hydrogen onone or more carbons of the alkynyl group. Such substituents may occur onone or more carbons that are included or not included in one or moretriple bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed elsewhere herein, except where stabilityis prohibitive. For example, substitution of alkynyl groups by one ormore alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

The term “amide” as used herein refers to a group —C(═O)N(R¹⁰)(R¹⁰)wherein each R¹⁰ is independently H or hydrocarbyl group, or two R¹⁰ aretaken together with the N atom to which they are attached to form aheterocycle having 4-8 atoms in the ring structure.

The terms “amine” and “amino” refer to both unsubstituted andsubstituted amines and salts thereof, e.g., a moiety that can berepresented by —N(R¹⁰)(R¹⁰) or —N⁺(R¹⁰)(R¹⁰)(R¹⁰), wherein each R¹⁰ isindependently H or a hydrocarbyl group, or two R¹⁰ are taken togetherwith the N atom to which they are attached to form a heterocycle having4-8 atoms in the ring structure.

The term “aminoalkyl” as used herein refers to an alkyl groupsubstituted with an amino group.

The term “APAP” refers to acetyl para-aminophenol.

The term “aralkyl” as used herein refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups that contain from x to y carbons in the chain. Forexample, the term “C_(x-y) alkyl” refers to substituted or unsubstitutedsaturated hydrocarbon groups, including straight-chain alkyl andbranched-chain alkyl groups that contain from x to y carbons in thechain, including haloalkyl groups such as trifluoromethyl and2,2,2-trifluoroethyl, and the like. C₀ alkyl indicates a hydrogen wherethe group is in a terminal position, a bond if internal. The terms“C_(2-y) alkenyl” and “C_(2-y) alkynyl” refer to substituted orunsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

The term “carbamate” refers to a group —OC(═O)NR⁹R¹⁰ or —N(R⁹)C(═O)OR¹⁰,wherein R⁹ and R¹⁰ are represent H or a hydrocarbyl group, such as analkyl group, or R⁹ and R¹⁰ are taken together with the interveningatom(s) to form a 4 heterocycle having 4-8 atoms in the ring structure.

The terms “carbocycle” and “carbocyclic” as used herein refers to asaturated or unsaturated ring in which each atom of the ring is carbon.The term carbocycle includes both aromatic carbocycles and non-aromaticcarbocycles. Non-aromatic carbocycles include both cycloalkane rings, inwhich all carbon atoms are saturated, and cycloalkene rings, whichcontain at least one double bond. “Carbocycle” includes 5-7 memberedmonocyclic and 8-12 membered bicyclic rings. Each ring of a bicycliccarbocycle may be selected from saturated, unsaturated and aromaticrings. Carbocycle includes bicyclic molecules in which one, two or threeor more atoms are shared between the two rings. The term “fusedcarbocycle” refers to a bicyclic carbocycle in which each of the ringsshares two adjacent atoms with the other ring. Each ring of a fusedcarbocycle may be selected from saturated, unsaturated and aromaticrings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, maybe fused to a saturated or unsaturated ring, e.g., cyclohexane,cyclopentane, or cyclohexene. Any combination of saturated, unsaturatedand aromatic bicyclic rings, as valence permits, is included in thedefinition of carbocyclic. Exemplary “carbocycles” include cyclopentane,cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene andadamantane. Exemplary fused carbocycles include decalin, naphthalene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane,4,5,6,7-tetrahydro-1H-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles”may be substituted at any one or more positions capable of bearing ahydrogen atom.

The term “carbocyclylalkyl” as used herein refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbonate” refers to a group —OC(═O)OR¹⁰, wherein R¹⁰ ishydrocarbyl.

The term “carboxy” as used herein refers to a group represented by theformula —CO₂H.

The term “cell” is used herein in its broadest sense in the art andrefers to a living body that is a structural unit of tissue of amulticellular organism, surrounded by a membrane structure whichseparates the contents of the cell from the surrounding environment, hasthe capability of self replicating, and has genetic information and amechanism for expressing it. Cells used herein may benaturally-occurring cells or artificially modified cells (e.g., fusioncells, genetically modified cells, etc.).

“A cell of the invention” or “cells of the invention” as used hereinmean a cell, cells, and/or a population of cells as described hereinand/or obtainable by methods described herein.

As used herein, “cellular differentiation” or “differentiation” is theprocess by which a less specialized cell becomes a more specialized celltype.

A “cycloalkyl” group is a cyclic hydrocarbon which is completelysaturated. “Cycloalkyl” includes monocyclic and bicyclic rings.Typically, a monocyclic cycloalkyl group has from 3 to about 10 carbonatoms, more typically 3-8 carbon atoms unless otherwise defined. Thesecond ring of a bicyclic cycloalkyl may be selected from saturated,unsaturated and aromatic rings. Cycloalkyl includes bicyclic moleculesin which one, two or three or more atoms are shared between the tworings. The term “fused cycloalkyl” refers to a bicyclic cycloalkyl inwhich each of the rings shares two adjacent atoms with the other ring.The second ring of a fused bicyclic cycloalkyl may be selected fromsaturated, unsaturated and aromatic rings. A “cycloalkenyl” group is acyclic hydrocarbon containing one or more double bonds.

The term “detect” refers to identifying the presence, absence, level, orconcentration of an analyte.

As used herein, the term “DOS 0” refers to BRD-K05085281; or 106.

As used herein, the term “DOS 1” refers to BRD-K17976466; or 108.

As used herein, the term “DOS 3” refers to BRD-K37628956; or 107.

“Embryonic stem (ES) cells” are pluripotent stem cells derived fromearly embryos. ES cells have been applied to production of knockoutmice, and human ES cells have been established and made available forregenerative medicine.

The term “ester,” as used herein, refers to a group —C(═O)OR¹⁰ whereinR¹⁰ is hydrocarbyl.

The term “ether” as used herein refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The term “hepatocyte” as used herein includes hepatocyte-like cells thatexhibit some but not all characteristics of mature hepatocytes, as wellas mature and fully functional hepatocytes.

The terms “hetaralkyl” and “heteroaralkyl” as used herein refers to analkyl group substituted with a heteroaryl group.

The term “heteroalkyl” as used herein refers to a saturated orunsaturated chain of carbon atoms and at least one heteroatom, whereinno two heteroatoms are adjacent, with the exception of N—O and N—N.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more atoms are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The terms “heterocyclyl,” “heterocycle” and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heterocyclyl” and “heterocyclic” also include polycyclic ring systemshaving two or more cyclic rings in which two or more atoms are common totwo adjoining rings wherein at least one of the rings is heterocyclic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclylgroups include, for example, piperidine, piperazine, pyrrolidine,morpholine, lactones, lactams, and the like.

The term “heterocyclylalkyl” as used herein refers to an alkyl groupsubstituted with a heterocycle group.

The term “hydrocarbyl” as used herein refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, heteroaryl, carbocycle,heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl” as used herein refers to an alkyl groupsubstituted with a hydroxy group.

The term “iHep” relating to cells refers to hepatocyte-like cells.

“Induced pluripotent stem cells,” commonly abbreviated as iPS cells oriPSCs, refer to a type of pluripotent stem cell artificially preparedfrom a non-pluripotent cell, typically an adult somatic cell, orterminally differentiated cell, such as fibroblast, a hematopoieticcell, a myocyte, a neuron, an epidermal cell, or the like, by insertingcertain genes, referred to as reprogramming factors.

As used herein, “isolated” refers to a molecule that is substantiallyfree of other elements present in its natural environment. For instance,an isolated protein is substantially free of cellular material or otherproteins from the cell or tissue source from which it is derived. Theterm “isolated” also refers to preparations where the isolated proteinis sufficiently pure to be administered as a pharmaceutical composition,or at least 70-80% (w/w) pure, more preferably, at least 80-90% (w/w)pure, even more preferably, 90-95% pure; and, most preferably, at least95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer non-hydrogen atoms in thesubstituent, preferably six or fewer. A “lower alkyl,” for example,refers to an alkyl group that contains ten or fewer carbon atoms,preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl,alkenyl, alkynyl, or alkoxy substituents defined herein are respectivelylower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, orlower alkoxy, whether they appear alone or in combination with othersubstituents, such as in the recitations hydroxyalkyl and aralkyl (inwhich case, for example, the atoms within the aryl group are not countedwhen counting the carbon atoms in the alkyl substituent).

In contrast, many progenitor cells are “multipotent stem cells,” i.e.,they are capable of differentiating into a limited number of cell fates.Multipotent progenitor cells can give rise to several other cell types,but those types are limited in number. An example of a multipotent stemcell is a hematopoietic cell—a blood stem cell that can develop intoseveral types of blood cells, but cannot develop into brain cells orother types of cells. At the end of the long series of cell divisionsthat form the embryo are cells that are terminally differentiated, orthat are considered to be permanently committed to a specific function.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

“Pluripotency” refers to a stem cell that has the potential todifferentiate into all cells constituting one or more tissues or organs,or preferably, any of the three germ layers: endoderm (interior stomachlining, gastrointestinal tract, the lungs), mesoderm (muscle, bone,blood, urogenital), or ectoderm (epidermal tissues and nervous system).“Pluripotent stem cells” used herein refer to cells that candifferentiate into cells derived from any of the three germ layers, forexample, direct descendants of totipotent stem cells or inducedpluripotent stem cells.

The terms “polycyclyl,” “polycycle” and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 5 to 7.

The term “prodrug” is intended to encompass compounds which, underphysiologic conditions, are converted into the therapeutically activeagents of the present invention (e.g., a compound of Formula (I)-(V) orof Table 1). A common method for making a prodrug is to include one ormore selected moieties which are hydrolyzed and/or metabolized underphysiologic conditions to reveal the desired molecule. In otherembodiments, the prodrug is converted by an enzymatic activity of thehost animal. For example, esters or carbonates (e.g., esters orcarbonates of alcohols or carboxylic acids) are prodrugs of the presentinvention. In certain embodiments, some or all of the compounds of theinvention can be replaced with a suitable corresponding prodrug, e.g.,wherein a hydroxyl in the parent compound is presented as an ester or acarbonate or carboxylic acid present in the parent compound is presentedas an ester.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

The term “salts” embraces addition salts of free acids that are usefulwithin the methods of the invention. The term “pharmaceuticallyacceptable salt” refers to salts that possess toxicity profiles within arange that affords utility in pharmaceutical applications.Pharmaceutically unacceptable salts may nonetheless possess propertiessuch as high crystallinity, which have utility in the practice of thepresent invention, such as for example utility in process of synthesis,purification or formulation of compounds useful within the methods ofthe invention. Suitable pharmaceutically acceptable acid addition saltsmay be prepared from an inorganic acid or from an organic acid. Examplesof inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric,carbonic, sulfuric (including sulfate and hydrogen sulfate), andphosphoric acids (including hydrogen phosphate and dihydrogenphosphate). Appropriate organic acids may be selected from aliphatic,cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic andsulfonic classes of organic acids, examples of which include formic,acetic, propionic, succinic, glycolic, gluconic, lactic, malic,tartaric, citric, ascorbic, glucuronic, maleic, malonic, saccharin,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, stearic, alginic, hydroxybutyric,salicylic, galactaric and galacturonic acid. Suitable pharmaceuticallyacceptable base addition salts of compounds of the invention include,for example, metallic salts including alkali metal, alkaline earth metaland transition metal salts such as, for example, calcium, magnesium,potassium, sodium and zinc salts. Pharmaceutically acceptable baseaddition salts also include organic salts made from basic amines suchas, for example, N,N′-dibenzylethylene-diamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocaine. All of these salts may be prepared from the correspondingcompound by reacting, for example, the appropriate acid or base with thecompound.

The term “silyl” refers to a silicon moiety with three hydrocarbylmoieties attached thereto.

As used herein, the term “somatic cell” refers to any cell other thangerm cells, such as an egg, a sperm, or the like, which does notdirectly transfer its DNA to the next generation. Typically, somaticcells have limited or no pluripotency. Somatic cells used herein may benaturally-occurring or genetically modified.

As used herein, the term “stem cell” refers to a cell capable of givingrise to at least one type of a more specialized cell. A stem cell hasthe ability to self-renew, i.e., to go through numerous cycles of celldivision while maintaining the undifferentiated state, and has potency,i.e., the capacity to differentiate into specialized cell types.Typically, stem cells can regenerate an injured tissue. Stem cellsherein may be, but are not limited to, embryonic stem (ES) cells,induced pluripotent stem cells, or tissue stem cells (also calledtissue-specific stem cells or somatic stem cells). Any artificiallyproduced cell which can have the above-described abilities (e.g., fusioncells, reprogrammed cells, or the like used herein) may be a stem cell.

Cells are “substantially free” of certain undesired cell types, as usedherein, when they have less that 10% of the undesired cell types, andare “essentially free” of certain cell types when they have less than 1%of the undesired cell types. However, even more desirable are cellpopulations wherein less than 0.5% or less than 0.1% of the total cellpopulation comprises the undesired cell types. Thus, cell populationswherein less than 0.1% to 1% (including all intermediate percentages) ofthe cells of the population comprise undesirable cell types areessentially free of these cell types. A medium may be “essentially free”of certain reagents, as used herein, when there is no external additionof such agents. More preferably, these agents are absent or present atan undetectable amount.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, andthe like. As used herein, the term “substituted” is contemplated toinclude all permissible substituents of organic compounds. In a broadaspect, the permissible substituents include acyclic and cyclic,branched and unbranched, carbocyclic and heterocyclic, aromatic andnon-aromatic substituents of organic compounds. The permissiblesubstituents can be one or more and the same or different forappropriate organic compounds. For purposes of this invention, theheteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. Substituents can include anysubstituents described herein, for example, a halogen, a hydroxyl, acarbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl),a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic moiety. It will be understood by those skilled in the artthat substituents can themselves be substituted, if appropriate. Unlessspecifically stated as “unsubstituted,” references to chemical moietiesherein are understood to include substituted variants. For example,reference to an “aryl” group or moiety implicitly includes bothsubstituted and unsubstituted variants.

The term “sulfate” refers to the group —OSO₃H, or a pharmaceuticallyacceptable salt thereof.

The term “sulfonamide” refers to the group represented by the generalformulae —S(═O)₂NR⁹N¹⁰ or —N(R⁹)S(═O)₂R¹⁰, wherein R⁹ and R¹⁰ areindependently H or hydrocarbyl, such as alkyl, or R⁹ and R¹⁰ takentogether with the intervening atom(s) complete a heterocycle having from4 to 8 atoms in the ring structure.

The term “sulfoxide” refers to the group —S(O)—R¹⁰, wherein R¹⁰ ishydrocarbyl.

The term “sulfonate refers to the group SO₃H, or a pharmaceuticallyacceptable salt thereof.

The term “sulfone” refers to the group —S(O)₂—R¹⁰, wherein R¹⁰ ishydrocarbyl.

The term “thioalkyl” as used herein refers to an alkyl group substitutedwith a thiol group.

The term “thioester” as used herein refers to a group —C(═O)SR¹⁰ or—SC(═O)R¹⁰ wherein R¹⁰ is hydrocarbyl.

The term “thioether” as used herein is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

Unlike ES cells, “tissue stem cells: have a limited differentiationpotential. Tissue stem cells are present at particular locations intissues and have an undifferentiated intracellular structure. Therefore,the pluripotency of tissue stem cells is typically low. Tissue stemcells have a higher nucleus/cytoplasm ratio and have few intracellularorganelles. Most tissue stem cells have low pluripotency, a long cellcycle, and proliferative ability beyond the life of the individual.Tissue stem cells are separated into categories, based on the sites fromwhich the cells are derived, such as the dermal system, the digestivesystem, the bone marrow system, the nervous system, and the like. Tissuestem cells in the dermal system include epidermal stem cells, hairfollicle stem cells, and the like. Tissue stem cells in the digestivesystem include pancreatic (common) stem cells, liver stem cells, and thelike. Tissue stem cells in the bone marrow system include hematopoieticstem cells, mesenchymal stem cells, and the like. Tissue stem cells inthe nervous system include neural stem cells, retinal stem cells, andthe like.

As used herein, “totipotent stem cells” refers to cells has the abilityto differentiate into all cells constituting an organism, such as cellsthat are produced from the fusion of an egg and sperm cell. Cellsproduced by the first few divisions of the fertilized egg are alsototipotent. These cells can differentiate into embryonic andextraembryonic cell types. Pluripotent stem cells can give rise to anyfetal or adult cell type. However, alone they cannot develop into afetal or adult animal because they lack the potential to contribute toextraembryonic tissue, such as the placenta.

The term “treating” comprises administration to the host of one or moreof the subject compositions, e.g., to diminish, ameliorate, or stabilizethe existing unwanted condition or side effects thereof.

The term “urea” is art-recognized and may be represented by the generalformula —N(R⁹)C(═O)NR⁹R¹⁰, wherein R⁹ and R¹⁰ are independently H orhydrocarbyl, such as alkyl, or either occurrence of R⁹ taken togetherwith R¹⁰ and the intervening atom(s) complete a heterocycle having 4-8atoms in the ring structure.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Compounds

The invention provides compounds or pharmaceutically acceptable saltsthereof, pharmaceutical compositions comprising the same, and methodsfor using the same.

In certain embodiments, the compound is a compound of Formula (I) or apharmaceutically acceptable salt thereof:

wherein in (I):W is H, F, Cl, Br, I, OH, lower alkyl, or lower alkoxy; X is F, Cl, Bror I; Y and Z are independently C(═O) or S(═O)₂; R¹ is lower alkyl; eachoccurrence of R² is independently selected from the group consisting ofH, OH, F, Cl, Br, I, alkyl, perfluoroalkyl (e.g., trifluoromethyl)),alkoxy, perfluoroalkoxy (e.g., trifluoromethoxy), NH₂, acylamino, amido,carboxyl, alkoxycarbonyl, acyloxy, formyl, acyl (includingperfluoroacyl, e.g., C(═O)CF₃)), thioester (e.g., thioacetate orthioformate), carbamate, urea, sulfonate, sulfamoyl, sulfone,sulfonamide, CN, NO₂, and alkylthio; and, n is 0, 1, 2, 3, 4 or 5.

In certain embodiments, n is 0, 1, 2, 3, 4 or 5, and (i) R₂ substituentoccupies the para position relative to NH group and is selected from thegroup comprising H, Cl, F, NH₂, and OH; preferably, R₂ substituent is H,or (ii) at least one non-hydrogen substituent is disposed ortho to theNH, preferably selected (independently, if non-hydrogen substituents arepresent at both ortho positions) from halogen (preferably fluorine),hydroxy, cyano, nitro, lower alkyl, lower alkoxy, and lower alkylthio,or (iii) both.

In certain embodiments, n is 0, 1, 2, 3, 4 or 5, and R₂ occupies thepara position relative to NH group and is selected from the groupcomprising H, Cl, F, NH₂, and OH; preferably, R₂ is H. In certainembodiments, n is 0, 1, 2, 3, 4 or 5, and at least one R₂ is not H andoccupies the ortho position relative to the NH group; preferably, atleast one R₂ is selected from the group consisting of halogen(preferably fluorine), hydroxy, cyano, nitro, lower alkyl, lower alkoxy,and lower alkylthio, and occupies the ortho position relative to the NHgroup.

In certain embodiments, n is 0, 1, 2, 3, 4 or 5, and (i) R₂ occupies thepara position relative to NH group and is selected from the groupcomprising H, Cl, F, NH₂, and OH; preferably, R₂ is H; and (ii) at leastone R₂ is not H and occupies the ortho position relative to the NHgroup; preferably, at least one R₂ is selected from the group consistingof halogen (preferably fluorine), hydroxy, cyano, nitro, lower alkyl,lower alkoxy, and lower alkylthio, and occupies the ortho positionrelative to the NH group.

In certain embodiments, Y is C(═O) and Z is S(═O)₂. In certainembodiments, X is chlorine and W is methyl. Non-limiting presentativecompounds of Formula (I) include FPH2 and compounds 102 and 103.

In certain embodiments, the compound is a compound of Formula (II) or apharmaceutically acceptable salt thereof:

wherein in (II):W is H, F, Cl, Br, I, OH, lower alkyl, or lower alkoxy; X is F, Cl, Bror I; each occurrence of R is independently H or lower alkyl; R³¹ is Hor lower alkyl; and R³² is amido, carboxyl, alkoxycarbonyl, orsulfonamide.

In certain embodiments, X is Cl and W is methyl. Non-limitingrepresentative compounds of Formula (II) include FPH1.

In certain embodiments, the compound is a compound of Formula (III) or apharmaceutically acceptable salt thereof:

wherein in (III):G is C(R)₂, O or N(R); each occurrence of Q is independently O or N(R);each occurrence of R is independently H or lower alkyl; each occurrenceof Y is independently C(═O) or S(O)₂; and R⁴¹ is lower alkyl or loweralkoxy.

In certain embodiments, the compound is symmetrical (i.e., bothoccurrences of Q, Y, and R⁴¹ are identical). In certain embodiments,both occurrences of Q are NH. In other embodiments, both occurrences ofY are C(═O). In yet other embodiments, both occurrences of R⁴¹ aremethyl. In yet other embodiments, both occurrences of Q are NH; bothoccurrences of Y are C(═O); and both occurrences of R⁴¹ are methyl. Incertain embodiments, each occurrence of Y is C(═O). Non-limitingrepresentative compounds of Formula (III) include FH1.

In certain embodiments, the compound is a compound of Formula (IV) or apharmaceutically acceptable salt thereof:

wherein in (IV):each occurrence of R is independently H or lower alkyl; Q is a bond, Oor N(R); Y is C(═O) or S(O)₂; R¹¹ is substituted or unsubstituted alkyl(including alkoxyalkyl and aminoalkyl), cycloalkyl, aryl, heteroaryl oraralkyl; R¹² is H or lower alkyl; R¹³ is H or substituted orunsubstituted lower alkyl (e.g., hydroxyalkyl); le is H, F, Cl, Br, I,substituted or unsubstituted aryl or heteroaryl, or —C≡C—R¹⁶, where R¹⁶is substituted or unsubstituted aminoalkyl, alkoxyalkyl, aryl orheteroaryl; each occurrence of R¹⁵ is independently OH, F, Cl. Br, I,NH₂, CN, NO₂, lower alkyl (such as perfluoroalkyl, e.g.,trifluoromethyl), or lower alkoxy; and, n is 0, 1 or 2.

In certain embodiments, Q is N(R). In other embodiments, Y is C(═O). Inyet other embodiments, R¹¹ is substituted or unsubstituted aryl, alkyl,alkoxyalkyl or heteroaryl. In yet other embodiments, R¹³ ishydroxyalkyl. Non-limiting representative compounds of Formula (IV)include 106-107, 201-205, 207, 209, 215, 223-224, 229-239, 243-244,248-249, 252, 254-255 and 257. In certain embodiments, Q is O, and R¹¹is substituted or unsubstituted aralkyl, alkyl, or alkoxyalkyl; and mostpreferably substituted or unsubstituted alkyl or alkoxyalkyl.

In certain embodiments, the substituents on the central eight-memberedring of a compound of Formula (IV) have the relative and/or absolutestereochemical orientation as shown for any one of the representativecompounds 106-107, 201-205, 207, 209, 215, 223-224, 229-239, 243-244,248-249, 252, 254-255 and 257.

In certain embodiments, the compound is a compound of Formula (V) or apharmaceutically acceptable salt thereof:

wherein in (V):each occurrence of R is independently H or lower alkyl; Q is a bond, Oor NR; Y is C═O or S(O)₂, preferably C═O; R²¹ is substituted orunsubstituted alkyl (including alkoxyalkyl and aminoalkyl), aralkyl,cycloalkyl, aryl or heteroaryl; R²² is H or lower alkyl; R²³ is H orsubstituted or unsubstituted lower alkyl (e.g., hydroxyalkyl); and R²⁵is H, OH, F, Cl, Br, I, lower alkyl (such as perfluoroalkyl, e.g.,trifluoromethyl), lower alkoxy, NH₂, CN, or NO₂.

In certain embodiments, Y is C═O. In other embodiments, R²¹ issubstituted or unsubstituted cycloalkyl, aralkyl, C₂-C₆ alkyl,aminoalkyl, aryl or heteroaryl. Non-limiting representative compounds ofFormula (V) include 108, 206, 208, 210-212, 217, 225-228, 240-242,245-247, 250-251, 253 and 256. In certain embodiments, Q is O, and R²¹is substituted or unsubstituted aralkyl, alkyl, or alkoxyalkyl; mostpreferably substituted or unsubstituted alkyl or alkoxyalkyl.

In certain embodiments, the substituents on the macrocycle of a compoundof Formula (V) have the absolute and/or relative stereochemicalorientation as shown for any one of the representative compounds 108,206, 208, 210-212, 217, 225-228, 240-242, 245-247, 250-251, 253 and 256.

Those of skill in the art will recognize that, with respect to Formulas(IV) and (V), there is significant structural homology among thesubstituents around the central ring and around the heterocyclic rings.Accordingly, activity relationships observed with respect to Q, Y, R,R¹¹, R¹², and R¹³ in the compounds of Formula (IV) herein may indicatesuitable substituents for Q, Y, R, R²¹, R²², and R²³ in compounds ofFormula (V), and vice versa.

In certain embodiments, the invention provides a compound of Table 1, ora pharmaceutically acceptable salt or prodrug thereof.

TABLE 1 com- pound no. structure 101 (FPH2)

2-(N-(5-chloro-2-methylphenyl)methylsulfonamido)-N-(2,6-difluorophenyl)acetamide 102

2-(N-(5-chloro-2-methylphenyl)methylsulfonamido)-N-(2-(methylthio)phenyl)acetamide 103

N-(4-bromo-3-methylphenyl)-2-(N-(5-chloro-2-methylphenyl)methylsulfonamido)acetamide 104 (FPH1)

4-(3-(5-chloro-2-methoxyphenyl)thioureido)-1-ethyl-1H-pyrazole-3-carboxamide 105 (FH1)

N,N′-(methylenebis(4,1-phenylene))diacetamide 106

1-(((2R,3S)-8-(benzofuran-2-yl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 107

3-(4-fluorophenyl)-1-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 108

N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide 109 (PH1)

4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one 110

(1R,4R)-4-((R)-1-aminoethyl)-N-(pyridin- 4-yl)cyclohexane-1-carboxamide111

(1R,4R)-4-((R)-1-aminoethyl)-N-(pyridin- 4-yl) cyclohexanecarboxamide112

1-((9H-carbazol-4-yl)oxy)-3-((2-(2- methoxyphenoxy)ethyl)amino)propan-2-ol 113

3-(5H-dibenzo[a,d][7]annulen-5-ylidene)- N,N-dimethylpropan-1-amine 114

2-(2-(1,3-dioxoisoindolin-2-yl)acetamido)acetic acid 201

1-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-isopropyl-1-methylurea 202

1-(((2R,3S)-8-((3,4-dimethoxyphenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 203

2-fluoro-N-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-N-methylbenzamide 204

1-(((2R,3S)-8-(3-cyclopentylprop-1-yn-1-yl)-5-((R)-1-hydroxy-propan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b] [1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 205

3-(4-fluorophenyl)-1-(((2R,3R)-8-((4-fluorophenyl)ethynyl)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)- 1-methylurea 206

N-(((4R,5R,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide 207

3-(4-fluorophenyl)-1-(((2R,3S)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-8-phenyl-3,4,5,6-tetrahydro-2H- pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 208

isobutyl (((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)(methyl)carbamate 209

1-(((2R,3S)-8-(4-cyanophenyl)-5-(R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 210

N-(((4S,5R,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide 211

N-(((4R,5S,Z)-7-((R)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide 212

N-(((4S,5R,Z)-7-((R)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide 215

N-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-2- methoxy-N-methylacetamide217

isopropyl (((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)(methyl)carbamate 224

3-(4-fluorophenyl)-1-(((2R,3S)-8-((3-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 225

4-(dimethylamino)-N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)- N-methylbutanamide 226

N-(((4S,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide 227

N-(((4S,5S,Z)-7-((R)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide 228

N-(((4R,5R,Z)-7-((R)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclopropanecarboxamide 229

1-(((2R,3S)-8-(cyclopropylethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 230

3-(4-fluorophenyl)-1-(((2R,3S)-8-((R)-3-hydroxybut-1-yn-1-yl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 231

1-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxy-propan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methyl-3-phenylurea 232

3-(4-fluorophenyl)-1-(((2S,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 233

3-(4-fluorophenyl)-1-(((2R,3R)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 234

3-(4-fluorophenyl)-1-(((2S,3R)-8-((4-fluorophenyl)ethynyl)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 235

3-(4-fluorophenyl)-1-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 236

3-(4-fluorophenyl)-1-(((2R,3S)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 237

1-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-methoxyphenyl)-1-methylurea 238

3-(4-fluorophenyl)-1-(((2S,3S)-8-((4-fluorophenyl)ethynyl)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 239

3-(4-fluorophenyl)-1-(((2S,3R)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 240

N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylcyclohexanecarboxamide 241

N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methyl-2-phenylacetamide 242

N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylbenzamide 243

1-(((2R,3S)-8-(3-(dimethylamino)prop-1-yn-1-yl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)- 3-(4-fluorophenyl)-1-methylurea244

1-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methyl-3-(naphthalen-1-yl)urea 245

N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N,1-dimethyl-1H-indole-6-carboxamide 246

N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methyl-5-phenylisoxazole-3-carboxamide 247

N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylisonicotinamide 248

3-(3,5-dimethylisoxazol-4-yl)-1-(((2R,3S)-8-((4-fluorophenyl)ethynyl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 249

3-(4-fluorophenyl)-1-(((2R,3S)-8-(3-hydroxyhex-1-yn-1-yl)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 250

benzyl (((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)(methyl)carbamate 251

ethyl (((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)(methyl)carbamate 252

3-(4-fluorophenyl)-1-(((2R,3S)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-8-(phenylethynyl)-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 253

1-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-3-isopropyl-1-methylurea 254

3-(4-fluorophenyl)-1-(((2R,3S)-5-((R)-1-hydroxypropan-2-yl)-3-methy1-6-oxo-8-((4-(trifluoromethyl)phenyl)ethynyl)-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea 255

1-(((2R,3S)-8-bromo-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-3-(4-fluorophenyl)-1-methylurea 256

4,4,4-trifluoro-N-(((4R,5S,Z)-7-((S)-1-hydroxypropan-2-yl)-5-methyl-8-oxo-11H-3-oxa-7-aza-1(4,1)-triazolacycloundecaphane-4-yl)methyl)-N-methylbutanamide 257

3-(4-fluorophenyl)-1-(((2R,3S)-5-((R)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-8-(3-(trifluoromethyl)phenyl)-3,4,5,6-tetrahydro-2H-pyrido[2,3-b][1,5]oxazocin-2-yl)methyl)-1-methylurea

In certain embodiments, compounds for inducing differentiation of cells,such as endodermal or mesodermal derived cells, comprise PH1, FH1, FPH1,FPH2, and compounds 106-108, 112-114, 201-212, 217 and 223-257. Incertain embodiments, compounds for inducing differentiation of cellscomprise FH1, FPH1, FPH2, and compounds 106, 108, 201-212, 217 and223-257. In certain embodiments, compounds for inducing proliferation ofcells, such as endodermal or mesodermal derived cells, include PH1,FPH1, FPH2, and compounds 102-103, 106-108, 110-112, 205, 211, 215,235-246, 248-250, 252, 255 and 257. In certain embodiments, compoundsfor inducing proliferation of cells, comprise PH1, FPH1, FPH2, andcompounds, 102, 106-108, 236, 238-240, 244, 248, 252 and 257.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising an effective amount of a compound of theinvention or a pharmaceutically acceptable salt or prodrug thereof, anda pharmaceutical carrier, diluent, or excipient.

In certain embodiments, the present invention provides a pharmaceuticalcomposition suitable for use in a human patient, comprising at least oneof the compounds illustrated herein (e.g., a compound of the invention,such as a compound of Table 1 or of Formulas (I)-(V)) and one or morepharmaceutically acceptable excipients. In other embodiments, thepharmaceutical preparations may be used in treating or preventing acondition or disease as described herein. In yet other embodiments, thepharmaceutical preparations have a low enough pyrogen activity to besuitable for administration into a human patient by, for example,injection.

Compounds of the invention can be prepared according to methods known inthe art. More specifically, compounds of formula (IV) can be preparedusing the retrosynthesis outlined in Scheme 1 for compound 106. Step 1comprises the coupling of compounds 1 and 2 to form compound 3. Forexample, compound 1 is reacted with compound 2 in the presence of PyBOPto generate the intermediate amide, which is then reduced to the amineusing BH₃-DMS, followed by quenching with Rochelle's salt in MeOH toform compound 3. Compounds 1 and 2 can be generated using reportedliterature procedures: Marcaurelle, L. A. et al., 2010, J. Am. Chem.Soc. 132:16962-16976. Step 2 comprises the acylation of compound 3 withcompound 14 to afford tertiary amide 9 in the presence of Et₃N. Step 3comprises the cyclization of compound 9 to form compound 10 in thepresence of CsF/NaH or TBAF. Step 4 comprises a manipulation of theamine protective group. Step 5 comprises the removal of the PMB groupusing DDQ to afford compound 29. Step 6 comprises the functionalizationof the compound 29. In one embodiment, compound 29 is loaded onto asolid support. For example, the solid support can besilicon-functionalized Lanterns. Compound 29 is deprotected and cappedwith the appropriate 4-fluorophenyl isocyanate to form the desired urea.Subsequent Suzuki cross-coupling with 2-benzofuranylboronic acid formscompound 106. In one aspect, the cleavage from the Lantern can beachieved by treatment with acid, such as 15% HF/pyridine in THF.

Compound 107 can be synthesized similar to the procedures employed forthe synthesis of compound 106. For example, in Step 6, compound 29 isfunctionalized with the desired functional groups to form compound 107.After the formation of the urea, identical to the synthesis of compound106, Sonogashira cross coupling with 4-fluorophenylacetylene givescompound 107. In one embodiment, the cleavage from the Lantern can beachieved by treatment with 15% HF/pyridine in THF.

Compound 108 can be prepared following the retro-synthesis of Scheme 2.Step 1 comprises the coupling of compounds 1 and 2 to form compound 3.Compound 1 is reacted with compound 2 in the presence of PyBOP togenerate the intermediate amide, which was then reduced to the amineusing BH₃-DMS, followed by quenching with Rochelle's salt in MeOH toform compound 3. Compounds 1 and 2 can be generated using reportedliterature procedures: Marcaurelle, L. A. et al., 2010, J. Am. Chem.Soc. 132:16962-16976. Step 2 comprises the formation of compound 19.Compound 3 is acylated with azido acid 18, and then deprotection of theTBS group produced the alcoholic intermediate. Propargylation of thealcoholic intermediate using NaHMDS in THF and DMF leads to compound 19.Step 3 comprises the cyclization of compound 19 using polystyrene-boundcopper catalyst, PS—CuPF₆, to form compound 7. Steps 4a and 4b compriseprotection manipulations. Step 4a comprises the removal of the Boc groupusing TBSOTf followed by HF-pyridine to afford the desired amine, whichis then protected as the Fmoc carbamate 28. Step 4b comprises theremoval of the PMB group using DDQ to afford compound 31. Step 5comprises the functionalization of the compound 31. In one embodiment,compound 31 is loaded onto a solid support. For example, the solidsupport can be silicon-functionalized Lanterns. The Fmoc group ofcompound 31 can be removed using 20% piperidine in DMF. The intermediateis then treated with cyclopropanecarboxylic acid to form compound 108.In one embodiment, the cleavage from the Lantern can be achieved bytreatment with 15% HF/pyridine in THF.

In certain embodiments, compounds of the invention may be prodrugs ofthe compounds of Table 1 and Formulas (I)-(V). A “prodrug” is an agentconverted into the parent drug in vivo. In one embodiment, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically active form of the compound. Inanother embodiment, a prodrug is enzymatically metabolized by one ormore steps or processes to the biologically, pharmaceutically ortherapeutically active form of the compound. For example, a hydroxyl inthe parent compound is presented as an ester or a carbonate in thepro-drug, or a carboxylic acid present in the parent compound ispresented as an ester in the pro-drug. In certain such embodiments, theprodrug is metabolized to the active parent compound in vivo (e.g., theester is hydrolyzed to the corresponding hydroxyl, or carboxylic acid).

In certain embodiments, sites on, for example, the aromatic ring portionof compounds of the invention are susceptible to various metabolicreactions. Incorporation of appropriate substituents on the aromaticring structures may reduce, minimize or eliminate this metabolicpathway. In certain embodiments, the appropriate substituent to decreaseor eliminate the susceptibility of the aromatic ring to metabolicreactions is, by way of example only, a deuterium, a halogen, or analkyl group.

Compounds described herein also include isotopically-labeled compoundswherein one or more atoms is replaced by an atom having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number usually found in nature. Examples of isotopes suitablefor inclusion in the compounds described herein include and are notlimited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³²P, and ³⁵S. In certain embodiments, isotopically-labeledcompounds are useful in drug and/or substrate tissue distributionstudies. In certain embodiments, substitution with heavier isotopes suchas deuterium affords greater metabolic stability (for example, increasedin vivo half-life or reduced dosage requirements). In certainembodiments, substitution with positron emitting isotopes, such as ¹¹C,¹⁸F, ¹⁵O and ¹³N, is useful in Positron Emission Topography (PET)studies for examining substrate receptor occupancy. Isotopically-labeledcompounds are prepared by any suitable method or by processes using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed.

In certain embodiments, the compounds described herein are labeled byother means, including, but not limited to, the use of chromophores orfluorescent moieties, bioluminescent labels, or chemiluminescent labels.

In certain embodiments, compounds of the invention may be racemic. Incertain embodiments, compounds of the invention may be enriched in oneenantiomer. For example, a compound of the invention may have greaterthan 30% ee, 40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee, or even 95%or greater ee. In certain embodiments, compounds of the invention mayhave more than one stereocenter. In certain such embodiments, compoundsof the invention may be enriched in one or more diastereomer. Forexample, a compound of the invention may have greater than 30% de, 40%de, 50% de, 60% de, 70% de, 80% de, 90% de, or even 95% or greater de.

In certain embodiments, the present invention relates to methods oftreatment with a compound of the invention, or a pharmaceuticallyacceptable salt thereof. In certain embodiments, the therapeuticpreparation may be enriched to provide predominantly one enantiomer of acompound (e.g., of Formula (IV) or (V)). An enantiomerically enrichedmixture may comprise, for example, at least 60 mole % of one enantiomer,or more preferably at least 75, 90, 95, or even 99 mole %. In certainembodiments, the compound enriched in one enantiomer is substantiallyfree of the other enantiomer, wherein substantially free means that thesubstance in question makes up less than 10%, or less than 5%, or lessthan 4%, or less than 3%, or less than 2%, or less than 1% as comparedto the amount of the other enantiomer, e.g., in the composition orcompound mixture. For example, if a composition or compound mixturecontains 98 grams of a first enantiomer and 2 grams of a secondenantiomer, it would be said to contain 98 mol percent of the firstenantiomer and only 2% of the second enantiomer.

In certain embodiments, the therapeutic preparation may be enriched toprovide predominantly one diastereomer of a compound (e.g., of Formula(IV) or (V)). A diastereomerically enriched mixture may comprise, forexample, at least 60 mole % of one diastereomer, or more preferably atleast 75, 90, 95, or even 99 mole %.

Cells and Cell Populations

The invention provides cells and populations of cells that may beobtained by methods described herein.

Methods of Expansion of Primary Cells

The invention provides a method for inducing proliferation of primarycells, comprising contacting the primary cells with a compound of theinvention, thereby inducing proliferation of the primary cells. Incertain embodiments, the resulting primary cells are differentiated. Themethod may be conducted in vitro or in vivo.

The invention provides a cell (or a population of cells) produced by amethod for inducing proliferation as described herein. The inventionfurther includes a cell (or a population of cells) produced by a methodfor inducing proliferation as described herein and characterized by oneor more of the assays described herein.

In certain embodiments, the primary cells comprise stem cells or aprogeny thereof. In certain embodiments, the stem cells are pluripotentor non-pluripotent. Exemplary pluripotent stem cells comprise inducedpluripotent stem cells, embryonic stem cells, and pluripotent stem cellsderived by nuclear transfer, cell fusion, or forced expression ofreprogramming factors. In certain embodiments, the stem cells areselected from multipotent stem cells, oligopotent stem cells, andunipotent stem cells. In certain embodiments, the stem cells comprisefetal stem cells or adult stem cells. In certain embodiments, the stemcells comprise epithelial stem cells; in other embodiments, the stemcells comprise endothelial stem cells. In certain embodiments, theprimary cells comprise somatic cells. In certain embodiments, theprimary cells are derived from endoderm or mesoderm. In certainembodiments, the primary cells comprise primary hepatocytes.

In certain embodiments, the differentiated cells produced by the methodsdescribed herein are characterized by one or more different assays,e.g., high content imaging and competitive ELISA. In certainembodiments, the differentiated cells produced by the methods describedherein are characterized by proliferation of normally quiescent cells(e.g., Ki67 staining). In certain embodiments, proliferation isdetectable by Ki67 via immunofluorescent staining (e.g., usingantibodies Ab15580 from Abcam, and AB9260 from Millipore). In certainembodiments, proliferation is 105%-infinite (if starting material hadzero Ki67-positive cells), such as 1,250%-1,900%.

In certain embodiments, the following functions are present in primarycells (prior to compound contact) and remain present in thedifferentiated cells produced by the methods described herein (aftercompound contact): albumin secretion, urea secretion, general cytochromeP450 activity, expression of mature liver genes, and bile transport(directional MRP-2 transport of CFDA as indicated by linear fluorescentregimes at the intercellular interface).

In certain embodiments, albumin secretion in the differentiated cells isdetectable via ELISA as compared to the primary hepatocytes. In otherembodiments, albumin secretion in the differentiated cells is 65%-130%changed as compared to the primary hepatocytes, e.g., 85%-110% changedas compared to primary hepatocytes.

In certain embodiments, urea secretion in the differentiated cells isdetectable via an assay kit (Stanbio Lab) as compared to the primaryhepatocytes. In other embodiments, urea secretion in the differentiatedcells is 80%-125% changed as compared to the primary hepatocytes, e.g.,95%-100% changed as compared to primary hepatocytes.

In certain embodiments, general cytochrome P450 activity in thedifferentiated cells is 105%-1,000% as compared to the primaryhepatocytes. In other embodiments, general cytochrome P450 activity inthe differentiated cells is 115%-730% changed as compared to the primaryhepatocytes, e.g., 125%-730% changed as compared to primary hepatocytes.

In certain embodiments, expression of mature liver genes in thedifferentiated cells is detectable via Luminex (Broad) as compared tothe primary hepatocytes. In other embodiments, expression of matureliver genes in the differentiated cells is 20%-820% changed as comparedto the primary hepatocytes, e.g., 20%-755% changed as compared toprimary hepatocytes.

In certain embodiments, bile transport (directional MRP-2 transport ofCFDA as indicated by linear fluorescent regimes at the intercellularinterface) in the differentiated cells is detectable by fluorescentimaging as compared to the primary hepatocytes. In other embodiments,bile transport in the differentiated cells is 80%-1,000% changed ascompared to the primary hepatocytes, e.g., 80%-120% changed as comparedto primary hepatocytes.

In certain embodiments, the increase in number of cell nuclei aftercontact with a compound of the invention is determined. In otherembodiments, the number of cell nuclei is increased in the population ofdifferentiated cells in comparison to the number of cell nuclei in theoriginal population of primary cells, e.g., by at least about 5-, 3-,1.5- or 0.5-fold.

In certain embodiments, the number of cell nuclei undergoing mitosisafter contact with a compound of the invention is determined. In otherembodiments, the number of cell nuclei undergoing mitosis is elevated inthe population of differentiated cells in comparison to the number ofcell nuclei undergoing mitosis in the original population of primarycells.

In certain embodiments, the extent of the proliferation is measured byone or more image-based readouts. In other embodiments, the imagereadouts are obtained by a process comprising quantifying the number ofnuclei in interphase, quantifying the number of nuclei in metaphase,and/or quantifying the number of nuclei in anaphase.

In certain embodiments, the level of secreted albumin as a marker forprotein synthesis after contact with a compound of the invention isdetermined. In other embodiments, the albumin secretion of thedifferentiated cells is increased at least about 2-fold in comparison tothe albumin secretion of the original population of primary cells.

In certain embodiments, the size of the colony of cells after contactwith a compound of the invention is determined. In other embodiments,the colony of differentiated cells is increased in size in comparison tothe colony size of the original population of primary cells. In yetother embodiments, the increase in colony size is at least about 4-fold,e.g., at least about 5-fold.

In certain embodiments, the phenotype of the cells is assessed aftercontact with a compound of the invention. In other embodiments, thedifferentiated cells produced comprise normal morphology. The phenotypeof the differentiated cells can be assessed using imaging, biochemicalanalyses, and gene expression profiling.

In certain embodiments, the phenotype of functional hepatocytes producedby methods described herein is assessed using imaging, biochemicalanalyses and gene expression profiling. In other embodiments, normalmorphology for functional hepatocytes produced by methods describedherein comprises a stable secretion of albumin at about 25 ug/10⁶heps/day. In yet other embodiments, normal morphology for functionalhepatocytes produced by methods described herein comprises a stablesynthesis of urea at about 150 ug/10⁶ heps/day. In yet otherembodiments, normal morphology for hepatocytes produced by methodsdescribed herein comprises cytochrome P450 activity at 0.3 uM/10⁶heps/hr. In yet other embodiments, normal morphology for hepatocytescomprises liver-specific gene expression.

Differentiation of iPS Cells

The invention provides a method for inducing the differentiation of apopulation of induced pluripotent stem cells (iPS cells). In certainembodiments, the method comprises contacting the population of iPS cellswith a compound of the invention, thereby inducing differentiation ofthe population of iPS cells into a population of differentiated cells.The method can be conducted in vitro or in vivo. Induced pluripotentstem cells (iPS cells) create the possibility of establishingpatient-specific cell types, thus empowering personal medicine. Theinvention provides a cell or a population of cells produced by a methodfor inducing the differentiation of induced pluripotent stem cells (iPScells) as described elsewhere herein. The invention further provides acell or a population of cells produced by the method for inducing thedifferentiation of induced pluripotent stem cells (iPS cells) asdescribed herein and characterized by one or more of the assaysdescribed herein.

In certain embodiments, the population of iPS cells prior to saiddifferentiation is a population of hepatocyte-like cells (iHep cells).In certain embodiments, the iHep cells express one or more fetalmarkers. In certain embodiments, the fetal marker comprises alphafetoprotein (AFP). In certain embodiments, the alpha fetoprotein isexpressed at a level detectable via immunofluorescent staining (e.g.,using antibody from Fitzgerald, 10-A05A).

In certain embodiments, the iHep cells prior to differentiation lackmature hepatocyte function. In certain embodiments, the maturehepatocyte function comprises CYP450 activity. In certain embodiments,the CYP450 activity is one or more selected from the group consisting ofCYP2A6 and CYP3A4 activity. In certain embodiments, the CYP2A6 activityis less than 0.1% of that found in mature hepatocytes. In certainembodiments, the CYP3A4 activity is less than 0.1% of that found inmature hepatocyte.

The iHep cells prior to differentiation may be produced by any suitablemethod. In certain embodiments, the iHep cells prior to saiddifferentiation are produced by (i) culturing undifferentiated iPS cellson Matrigel®; (ii) transferring confluent iPS cells to differentiationmedia; and (iii) adding growth factors (Activin A, BMP-4, bFGF, HGF, andOSM).

In certain embodiments, the methods comprise treating a population ofiHep cells with a compound of the invention. In certain embodiments, thepopulation of iHep cells is treated with a compound of the invention onat least one of day 21-35 post differentiation. The population of iHepcells may be treated with a compound of the invention on day 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 after saiddifferentiation. In certain embodiments, the population of iHep cells iscultured with a compound of the invention for a period of at least 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 days, e.g., at least 5 days.

In certain embodiments, the population of differentiated cells producedby the methods described herein is characterized by one or moredifferent assays. In certain embodiments, the population ofdifferentiated functional cells produced by the methods described hereinis characterized by an up regulation of CYP3A4 activity and a downregulation of AFP as compared to iPS-derived hepatocyte like cells (asdefined by Si-Tayeb K. et al., 2010, Hepatology 51:297-305,doi:10.1002/hep.23354 (“Si-Tayeb et al.”)). In certain embodiments, thepopulation of differentiated cells is characterized by an increase inone or more of the following functions: albumin secretion, intracellularalbumin stain, general CYP450 activity, expression of mature livergenes, CYP 2A6 activity, CYP 3A4 activity, CYP 3A stain, albumin andCYP3A double positive staining.

In certain embodiments, the population of differentiated cells ischaracterized by an increase in albumin secretion as compared toiPS-derived hepatocyte like cells (as defined by Si-Tayeb et al.). Incertain such embodiments, the increase is 100%-2,000%, e.g., theincrease is 100%-1650%, or even 170%-1,185%.

In certain embodiments, the population of differentiated cells ischaracterized by an increase in intracellular albumin stain as comparedto iPS-derived hepatocyte like cells (as defined by Si-Tayeb et al.). Incertain such embodiments, the increase is detectable viaimmunofluorescent staining (e.g., using antibody from BethylLaboratories, A80-229A). In certain such embodiments, the increase is100%-10,000%, or even 200%-6,975%.

In certain embodiments, the population of differentiated cells ischaracterized by an increase in general CYP450 activity as compared toiPS-derived hepatocyte like cells (as defined by Si-Tayeb et al.). Incertain such embodiments, the increase is 100%-150%, e.g., 105%-150%, oreven 130%-145%.

In certain embodiments, the population of differentiated cells ischaracterized by an increase in expression of mature liver genes ascompared to iPS-derived hepatocyte like cells (as defined by Si-Tayeb etal.). In certain such embodiments, the increase is 100%-33,335%, e.g.,100%-780%, or even 100%-675%.

In certain embodiments, the population of differentiated cells ischaracterized by an increase in CYP 2A6 activity as compared toiPS-derived hepatocyte like cells (as defined by Si-Tayeb et al.). Incertain embodiments, the increase is 105%-100,000%, such as 900%-7,270%,e.g., 2,330%-3,335%.

In certain embodiments, the population of differentiated cells ischaracterized by an increase in CYP 3A4 activity as compared toiPS-derived hepatocyte like cells (as defined by Si-Tayeb et al.). Incertain embodiments, the increase is 105%-100,000%, e.g., 300%-5,250%,such as 1,600%-4,590%.

In certain embodiments, the population of differentiated cells ischaracterized by an increase in albumin and CYP3A double positivestaining as compared to iPS-derived hepatocyte like cells (as defined bySi-Tayeb et al.). In certain embodiments, the increase is detectable viaimmunofluorescent staining (e.g., using CYP3A antibody from Santa Cruzsc271033, and albumin antibody from Bethyl Laboratories A80-229A. Incertain embodiments, the increase is 105%-100,000%, such as3640%-3,750%.

In certain embodiments, the population of differentiated cells ischaracterized by a decrease in alpha-fetoprotein secretion as comparedto iPS-derived hepatocyte like cells (as defined by Si-Tayeb et al.). Incertain embodiments, the decrease is 0.0%-95%, such as 8.0%-30%, e.g.,8.5%-20%.

In certain embodiments, the population of differentiated cells ischaracterized by a decrease in AFP stain as compared to iPS-derivedhepatocyte like cells (as defined by Si-Tayeb et al). In certainembodiments, the decrease is 0.0%-95% (e.g., using antibody fromFitzgerald, 10-A05A), such as 0.0%-50%, or even 0.0%-2.0%.

In certain embodiments, the population of differentiated cells producedby the method for inducing the differentiation described hereincomprises cells that are increased in size as compared to iPS-derivedhepatocyte like cells (as defined by Si-Tayeb et al.). In certainembodiments, the population of differentiated cells comprises cells withmore pronounced hepatocyte morphologies and with more noticeable bilecannaliculi transport between hepatocytes. In certain embodiments, thepopulation of differentiated cells comprises cells having CYP3A4activity at >0.1% of that found in mature hepatocytes. In certainembodiments, the population of differentiated cells comprises cellshaving GSTP1 expression at <60% of that found in undifferentiated iPScells. In certain embodiments, the population of differentiated cellscomprises cells having an AFP protein level at <40% of AFP levels foundin hepatocyte-like cells, as defined by Si-Tayeb et al. In certainembodiments, the population of differentiated cells comprises cellshaving albumin protein level at >1.5 fold of albumin levels found inhepatocyte-like cells, as defined by Si-Tayeb et al. In certainembodiments, the population of differentiated cells comprises cellshaving a CYP3A4 protein level at >5 fold of CYP3A4 levels found inhepatocyte-like cells, as defined by Si-Tayeb et al.

Methods of Use

Compounds of the invention can be used to grow and differentiate cells,thereby producing differentiated cells for use in a variety of in vitroand in vivo applications. A compound of the invention may be useful inany methods and applications that utilize such cells. Furthermore, acell of the invention (or a population of cells of the invention, i.e.,differentiated cells obtainable by the methods disclosed herein) mayalso be useful in any methods and applications that employ such cells.In certain embodiments, the cells for use in the methods for inducingproliferation of primary cells and the methods for inducingdifferentiation of a population of cells described herein may be stemcells or a progeny cell thereof. The stem cells may be pluripotent stemcells or any non-pluripotent stem cells. The pluripotent stem cells maybe induced pluripotent stem cells, embryonic stem cells, or pluripotentstem cells derived by nuclear transfer or cell fusion. The stem cellsmay also be multipotent stem cells, oligopotent stem cells, or unipotentstem cells. The stem cells may be fetal stem cells or adult stem cells,for example, epithelial stem cells or endothelial stem cells. In certainembodiments, the cells may be somatic cells, either immortalized or not.In certain embodiments, the cells are derived from endoderm or mesoderm.The cells may also be hepatocytes, more particularly, mature hepatocytesor immature hepatocytes (e.g., hepatocyte-like cells).

Compounds of the invention can be used to grow and differentiatehepatocytes. A compound of the invention and/or cells obtainable by themethods described herein may be useful in methods and applications thatutilize hepatocytes. For example, hepatocytes are used in methods ofassessing a pharmaceutical compound, comprising assaying apharmacological or toxicological property of the pharmaceutical compoundon hepatocytes or a tissue engineered liver. Additional uses ofhepatocytes include, but are not limited to, transplantation orimplantation of the hepatocytes in vivo; screening cytotoxic compounds,carcinogens, mutagens, growth/regulatory factors, pharmaceuticalcompounds, and the like, in vitro; elucidating the mechanism of liverdiseases and infections; studying the mechanism by which drugs and/orgrowth factors operate; diagnosing and monitoring cancer in a patient;gene therapy; and the production of biologically active products, toname but a few.

Additional uses for compounds of the invention and/or cells of theinvention may be for distribution for commercial, therapeutic, andresearch purposes. For purposes of manufacture, distribution, and use,the cells of the invention may be supplied in the form of a cell cultureor suspension in an isotonic excipient or culture medium, optionallyfrozen to facilitate transportation or storage. Compounds of theinvention may be useful for producing cells that may be used in methodsof screening e.g., drug testing, toxicity assays, factors that promotematuration, proliferation, and/or maintenance of cells in culture.Compounds of the invention and/or cells of the invention may also beused in numerous therapies and medical treatments. Compounds of theinvention and/or cells of the invention may be used in research, e.g.,to elucidate cellular growth mechanisms leading to the identification ofnovel targets for cancer therapies, to elucidate mechanisms involved incell fate determination leading to new strategies for cellularreprogramming, and to generate genotype-specific cells for diseasemodeling, including the generation of new therapies customized todifferent genotypes. Such customization can reduce adverse drug effectsand help identify therapies appropriate to the patient's genotype.

Test Compound Screening

Compounds of the invention can be used to produce cells that can be usedto screen for factors (such as solvents, small molecule drugs, peptides,and polynucleotides) or environmental conditions (such as cultureconditions or manipulation) that affect the characteristics of cells.

In one aspect, the invention provides a method of assessing a compoundfor a pharmacological or toxicological effect on a population ofdifferentiated cells, comprising: (a) contacting a population of primarycells or a population of induced pluripotent stem cells with a firstcompound of the invention to produce a population of differentiatedcells; (b) contacting the population of differentiated cells with a testagent, and (c) assaying for a pharmacological or toxicological effect ofthe test compound on said population. In certain embodiments, the cellscomprise hepatocytes.

In certain embodiments, a compound of the invention can be used toproduce hepatocytes that can be used to screen for factors (such assolvents, small molecule drugs, peptides, and polynucleotides) orenvironmental conditions (such as culture conditions or manipulation)that affect the characteristics of hepatocytes. Over the past decade, invitro models have been established, such as precision-cut liver slices,primary hepatocytes, and liver cell lines and a few studies examiningthe relevance of drug testing with hepatocyte cell lines, found thatcell lines poorly reproduce and predict drug metabolism andhepatotoxicity as opposed to primary hepatocytes or liver slices. Thus,primary human hepatocytes are the “gold standard” for in vitro drugtesting. However, the limited supply of human hepatocytes and the factthat such hepatocytes may not adequately represent the genetic variationof the patient population limit the ability to detect all potential drugtoxicities. Consequently, utilization of cells derived from a diversegroup of donors, e.g., by obtaining stem cells from a diverse group ofdonors (for instance by generating iPS cells) and differentiating themto hepatocytes (e.g., with differing cytochrome P450 profiles) wouldallow drug testing to more closely examine and predict potentialproblems for particular groups or individuals.

In some aspects, compounds of the invention are used to produce(un)differentiated stem cells that are used to screen factors thatpromote maturation of cells along the hepatocyte lineage, or promoteproliferation and maintenance of such cells in long-term culture. Forexample, candidate hepatocyte maturation factors or growth factors aretested by adding them to stem cells in different wells, and thendetermining any phenotypic change that results, according to desirablecriteria for further culture and use of the cells.

Particular screening applications of this invention relate to thetesting of pharmaceutical compounds in drug research (see In vitroMethods in Pharmaceutical Research, Academic Press, 1997, and U.S. Pat.No. 5,030,015). In certain aspects of this invention, compounds of theinvention are used to grow and differentiate hepatocytes to serve astest cells for standard drug screening and toxicity assays, as have beenpreviously performed on hepatocyte cell lines or primary hepatocytes inshort-term culture. Assessment of the activity of candidatepharmaceutical compounds generally involves contacting the hepatocytesproduced by one or more compounds of the invention with the candidatecompound, determining any change in the morphology, marker phenotype, ormetabolic activity of the cells that is attributable to the candidatecompound (e.g., by comparing the effects with control cells, such asuntreated cells or cells treated with an inert compound or vehicle), andthen correlating the effect of the candidate compound with the observedchange. The screening may be done either because the candidate compoundis being tested to see if it has a pharmacological effect on liver cells(e.g., a therapeutic effect), or because a candidate compound designedto have effects elsewhere may have unintended hepatic side effects. Twoor more drugs can be tested in combination (by combining with the cellseither simultaneously or sequentially) to detect possible drug-druginteraction effects.

In some applications, hepatocytes produced by one or more compounds ofthe invention are used to screen pharmaceutical compounds for potentialhepatotoxicity (Castell et al., In: In vitro Methods in PharmaceuticalResearch, Academic Press, 375-410, 1997. Cell Encapsulation Technologyand Therapeutics, Kuhtreiber et al. eds., Birkhauser, Boston, Mass.,1999). Cytotoxicity can be determined in the first instance by theeffect on cell viability, morphology, and leakage of enzymes into theculture medium. More detailed analysis is conducted to determine whetherthe pharmaceutical compounds affect cell function (such asgluconeogenesis, ureogenesis, and plasma protein synthesis) withoutcausing toxicity. Lactate dehydrogenase (LDH) is a good marker becausethe hepatic isoenzyme (type V) is stable in culture conditions, allowingreproducible measurements in culture supernatants after 12-24 hincubation. Leakage of enzymes such as mitochondrial glutamateoxaloacetate transaminase and glutamate pyruvate transaminase can alsobe used. Gomez-Lechon et al., Anal. Biochem., 236:296, 1996 describes amicroassay for measuring glycogen, which can be used to measure theeffect of pharmaceutical compounds on hepatocyte gluconeogenesis.

Other markers/functions useful to evaluate hepatotoxicity includedetermination of the synthesis and secretion of albumin, cholesterol,and lipoproteins; transport of conjugated bile acids and bilirubin;ureagenesis; cytochrome p450 levels and activities; glutathione levels;release of alpha-glutathione s-transferase; ATP, ADP, and AMPmetabolism; intracellular K⁺ and Ca²⁺ concentrations; the release ofnuclear matrix proteins or oligonucleosomes; and induction of apoptosis(indicated by cell rounding, condensation of chromatin, and nuclearfragmentation). DNA synthesis can be measured as [³H]-thymidine or BrdUincorporation. Effects of a drug on DNA synthesis or structure can bedetermined by measuring DNA synthesis or repair. [³H]-thymidine or BrdUincorporation, especially at unscheduled times in the cell cycle, orabove the level required for cell replication, is consistent with a drugeffect. Unwanted effects can also include unusual rates of sisterchromatid exchange, determined by metaphase spread. See Vickers in Invitro Methods in Pharmaceutical Research, Academic Press, 375-410, 1997for further elaboration.

Research Tools

In certain embodiments, the invention provides a method of developing ahumanized mouse model comprising administering to the mouse (i) humancells obtained by any of the methods described herein; (ii) one or morecells as described herein; or (iii) a combination thereof.

In certain embodiments, the invention further provides administering tothe mouse an effective amount of one or more compounds of invention, ora pharmaceutically acceptable salt or prodrug thereof. For example, amouse receiving human liver cells or tissue may also be treated with acompound of the invention to induce proliferation and/or differentiationof cells in the implanted material. Similarly, human liver cells ortissue may be grown ex vivo from human liver cells using the methodsdescribed herein or using cells as described herein, optionally throughthe use of one or more compounds of the invention to proliferate and/ordifferentiate the cells. In certain such embodiments, the tissue isgrown, at least in part, from cells obtained from the subject (i.e.,autologous cells). The mouse model may be used for the development of anew therapy, e.g., for liver disease.

Methods of Treatment

Compounds of the invention and/or cells of the invention are useful inmethods of treating, alleviating, and/or preventing a disease orcondition, such as a liver disease or condition, in a subject in needthereof. In certain embodiments, compounds of the invention and/or cellsof the invention are useful in methods for treating a subject having orat risk of a liver dysfunction. In certain embodiments, cells of theinvention can enable personalized medicine. For example,patient-specific cells can be obtained by using one or more compounds ofthe invention to expand patient biopsies and/or to differentiate patientiPS cells.

In certain embodiments, the invention provides a method of treating,alleviating, and/or preventing a disease or condition in a subject, suchas a human, comprising administering to a subject in need thereof (i) aneffective amount of one or more compounds of the invention; (ii) one ormore cells obtained by any of the methods described herein; or (iii) oneor more cells described herein; or (iv) a combination thereof.

In certain embodiments, the invention provides a method wherein acompound of the invention induces proliferation of endogenous cells. Incertain embodiments, the invention provides a method, wherein a compoundof the invention promotes differentiation of endogenous cells, e.g.,toward a more mature phenotype.

In certain embodiments, the endogenous cells are stem cells or a progenycell thereof. In certain embodiments, the stem cells are pluripotent ornon-pluripotent. Representative pluripotent stem cells include inducedpluripotent stem cells, embryonic stem cells, and pluripotent stem cellsderived by nuclear transfer, cell fusion, or forced expression ofreprogramming factors. In certain embodiments, the stem cells areselected from multipotent stem cells, oligopotent stem cells, andunipotent stem cells. In certain embodiments, the stem cells are fetalstem cells or adult stem cells. In certain embodiments, the stem cellsare epithelial stem cells; in others, endothelial stem cells. In certainembodiments, the cells are somatic cells. In certain embodiments, thecells are derived from endoderm or mesoderm. In certain embodiments, thecells are hepatocytes.

In certain embodiments, the invention provides a method of treating adisease or condition in a subject. In certain embodiments, the inventionprovides a method of alleviating a disease or condition in a subject. Incertain embodiments, the invention provides a method of preventing adisease or condition in a subject.

In certain embodiments, the disease or condition is a liver disease orcondition, e.g., acetaminophen toxicity, alcoholic liver disease,primary liver cancer, liver cirrhosis, liver cysts, fatty liver disease,liver fibrosis, hepatitis, primary sclerosing cholangitis, and jaundice.In certain embodiments, the method further comprises implanting into thesubject a bio-artificial liver device. In certain embodiments, thedisease is hepatitis, e.g., caused by a virus selected from hepatitis Avirus, hepatitis B virus, hepatitis C virus, hepatitis D virus, orhepatitis E virus, herpes simplex, cytomegalovirus, Epstein-Barr virus,and yellow fever.

In certain embodiments, the compound is administered by a route selectedfrom the group consisting of oral, parenteral, intramuscular,intranasal, sublingual, intratracheal, inhalation, ocular, vaginal,rectal, and intracerebroventricular. In certain embodiments, theadministration comprises oral.

In certain embodiments, the invention contemplates that the compound orcells being administered are encapsulated, such as in a nanoparticle orhydrogel.

The invention also includes a method for transplanting an organ ortissue into a subject in need thereof, comprising administering to thesubject (i) an effective amount of one or more compounds of theinvention; (ii) one or more cells obtained by any of the methodsdescribed herein; (iii) one or more cells described herein; or (iv) acombination thereof.

In certain embodiments, the invention provides a method fortransplanting an organ, such as a liver transplant. For example, apatient receiving an organ transplant may also be treated with acompound of the invention to induce proliferation and/or differentiationof cells in the transplanted organ. Similarly, the transplanted organmay be an organ grown ex vivo from cells obtained by the methodsdescribed herein or using cells as described herein, optionally throughthe use of one or more compounds of the invention to proliferate and/ordifferentiate the cells. In other embodiments, the organ is grown, atleast in part, from cells obtained from the subject (i.e., autologouscells).

In certain embodiments, the invention provides a method fortransplanting tissue. In certain embodiments, the tissue comprises livertissue. For example, a patient receiving a tissue transplant may also betreated with a compound of the invention to induce proliferation and/ordifferentiation of cells in the transplanted tissue. Similarly, thetransplanted tissue may be tissue grown ex vivo from cells obtained bythe methods described herein or using cells as described herein,optionally through the use of one or more compounds of the invention toproliferate and/or differentiate the cells. In other embodiments, thetissue is grown, at least in part, from cells obtained from the subject(i.e., autologous cells).

In certain embodiments, the invention provides a method for the repairof damaged, diseased, or aged tissue, such as liver tissue, in a subjectcomprising administering to a subject in need thereof (i) an effectiveamount of one or more compounds of the invention; (ii) one or more cellsobtained by any of the methods described herein; (iii) one or more cellsdescribed herein; or (iv) a combination thereof.

In certain embodiments, the compound is administered systemically orlocally. In certain embodiments, the compound or cells are administeredprior to an organ or tissue transplant. In certain embodiments, thecompound or cells are administered during an organ or tissue transplant.In certain embodiments, the compound or cells are administered after anorgan or tissue transplant.

In certain embodiments, the invention provides a method of treating asubject in need cell replacement therapy, comprising: providing apopulation of cells; inducing differentiation of said population ofcells by exposing said cells to a compound of the invention to produce apopulation of differentiated cells (e.g., hepatic cells); andadministering the population of differentiated cells to said subject inneed thereof. In certain embodiments, the population of cells is derivedfrom the subject in need of cell replacement therapy. In certainembodiments, the compound induces proliferation of the cells. In certainembodiments, the compound promotes differentiation of the cells.

Liver Therapy and Transplantation

The invention also provides methods of using compounds of the inventionand/or cells of the invention to restore a degree of liver function to asubject needing such therapy, e.g., due to an acute, chronic, orinherited impairment of liver function.

One of the unique features of the liver is its enormous naturalregeneration ability. This regeneration is due in large part to there-entry of terminally differentiated hepatocytes in the cell cycle,resulting in multiple cell divisions to regenerate the liver. When thehepatocytes are damaged, liver stem/progenitor cells, termed oval cellsin rodents, located in the peri-portal zone, are activated anddifferentiate to mature hepatocytes. Hence, most liver diseases endingin liver failure are caused by a combination of decreased proliferationof hepatocytes and exhaustion of the stem/progenitor cell pool. Liverfailure is caused by a number of disorders, including cirrhosis due toinfections, excessive alcohol consumption, genetic and idiopaticreasons. In addition, acute liver failure is caused by ingestion ofcertain drugs or foods. Liver transplantation is the only successfultreatment for end stage liver disease, and is in many instances also theonly curative therapy for certain forms of genetic disorders of theliver. Many liver disorders treated by whole liver transplantationresult from hepatocyte dysfunction. Hepatocyte transplantation is ofgreat interest. There is significant evidence that grafted hepatocytescan assume the full range of liver functions in vivo. Hepatocytetransplantation has several advantages over whole liver transplant:lower morbidity, a single donor organ can be used for severalrecipients, cells can be cryopreserved, and cells grafts are lessimmunogenic than whole organ grafts. However, lack of donor cellscurtails further exploration of this therapy. As a consequence, there isalso great interest in therapies to promote the growth anddifferentiation of hepatocytes for the treatment of acute and chronicliver failure, as well as inherited metabolic disorders. Compounds ofthe invention and/or cells of the invention may be useful in thetherapies described above.

Accordingly the invention is also directed to methods of treating liverdeficiencies by administering a compound of the invention and/or cellsof the invention to a subject with the liver deficiency. Suchdeficiencies include, but are not limited to, toxic liver disease,metabolic liver disease, acute liver necrosis, effects of acetaminophen,hemochromatosis, Wilson's Disease, Crigler Najar, hereditarytyrosinemia, familial intrahepatic cholestatis type 3, ornithinetranscarbamylase (OTC) deficiency, and urea cycle disorder.

Further diseases include, but are not limited to viral hepatitis,chronic viral hepatitis A, B, C, acute hepatitis A, B, C, D, E,cytomegalovirus and herpes simplex virus; liver dysfunction in otherinfectious diseases such as, without limitation, toxoplasmosis,hepatosplenic schistosomiasis, liver disease in syphilis, leptospirosisand amoebiasis; metabolic diseases such as, without limitation,haemochromatosis, Gilbert's syndrome, Dubin-Johnson syndrome and Rotor'ssyndrome; alcoholic liver disease such as, without limitation, fattyliver, fibrosis, sclerosis and cirrhosis; and toxic liver disease.

Compounds of the invention and/or cells of the invention may be assessedfor their ability to restore liver function in an animal lacking fullliver function. Compounds of the invention and/or cells of the inventionmay be assessed for their ability to restore liver function in an animalwith a humanized liver. For models of liver disease or failure, see:Braun et al., 2000, Nature Med., 6:320; Rhim et al., 1995, Proc. Natl.Acad. Sci. USA 92:4942; Lieber et al., 1995, Proc. Natl. Acad. Sci. USA92:6210; Mignon et al., 1998, Nature Med., 4:1185; Overturf et al.,1009, Human Gene Ther., 9:295. Acute liver disease can be modeled by 90%hepatectomy or by treating animals with a hepatotoxin such asgalactosamine, CCl₄, or thioacetamide.

Chronic liver diseases such as cirrhosis can be modeled by treatinganimals with a sub-lethal dose of a hepatotoxin long enough to inducefibrosis (Rudolph et al., 2000, Science 287:1253). Assessing the abilityof compounds of the invention and/or the cells of the invention toreconstitute liver function involves administering the compounds and/orcells to such animals, and then determining survival over a 1 to 8 weekperiod or more, while monitoring the animals for progress of thecondition. Effects on hepatic function can be determined by evaluatingmarkers expressed in liver tissue, cytochrome P450 activity, and bloodindicators, such as alkaline phosphatase activity, bilirubinconjugation, and prothrombin time), and survival of the host. Anyimprovement in survival, disease progression, or maintenance of hepaticfunction according to any of these criteria relates to effectiveness ofthe therapy.

Compounds of the invention may be administered orally. For purposes ofhemostasis, the compounds of the invention can be administered at anysite that has adequate access to the circulation, typically within theabdominal cavity. For some metabolic and detoxification functions, it isadvantageous for the compounds of the invention to have access to thebiliary tract. Accordingly, in certain embodiments, a compound of theinvention is administered locally, e.g., near the liver (e.g., in thetreatment of chronic liver disease) or the spleen (e.g., in thetreatment of fulminant hepatic failure). In certain embodiments, acompound of the invention is administered into the hepatic circulationeither through the hepatic artery, or through the portal vein, byinfusion through an in-dwelling catheter. A catheter in the portal veincan be manipulated so that the cells flow principally into the spleen,or the liver, or a combination of both. In certain embodiments, acompound of the invention is administered by placing a bolus in a cavitynear the target organ, typically in an excipient or matrix that willkeep the bolus in place. In certain embodiments, a compound of theinvention is injected directly into a lobe of the liver or the spleen.

A compound of the invention or cells of the invention can be used fortherapy of any subject in need of having hepatic function restored orsupplemented. Human conditions that may be appropriate for such therapyinclude fulminant hepatic failure due to any cause, viral hepatitis,drug-induced liver injury, cirrhosis, inherited hepatic insufficiency(such as Wilson's disease, Gilbert's syndrome, or alpha₁-antitrypsindeficiency), hepatobiliary carcinoma, autoimmune liver disease (such asautoimmune chronic hepatitis or primary biliary cirrhosis), and anyother condition that results in impaired hepatic function.

Use in a Liver Assist Device

Certain aspects of this invention include a compound of the inventionthat is encapsulated or part of a bioartificial liver device. Certainaspects of this invention include cells of the invention that areencapsulated or part of a bioartificial liver device. In another aspect,a compound of the invention is administered to a subject with abioartificial liver (BAL) device. In another aspect, a compound of theinvention is used to produce hepatocytes that can be encapsulatedaccording to such methods for use either in vitro or in vivo.

Bioartificial organs for clinical use are designed to support anindividual with impaired liver function, either as a part of long-termtherapy, or to bridge the time between a fulminant hepatic failure andhepatic reconstitution or liver transplant. See Macdonald et al., pp.252-286 of Cell Encapsulation Technology and Therapeutics, Kuhtreiber etal. eds., Birkhauser, Boston, Mass., 1999, and exemplified in U.S. Pat.Nos. 5,290,684, 5,624,840, 5,837,234, 5,853,717, and 5,935,849.

BAL devices are designed to support the detoxification functionsperformed by the liver, hence decreasing the risk and severity of CNScomplications associated with acute liver failure. It is also noted thatcompounds of the invention and/or cells of the invention may be used inconjunction with a BAL device to replace other liver functions. BALdevices could benefit three groups of patients: with fulminant hepaticfailure, waiting for an imminent transplant, and with early failure of aliver transplant. Although some positive results have been seen inpatients with liver failure, further exploration of the usefulness ofBAL devices has been hampered by lack of suitable cells. Currently,tumor-derived cell lines or animal cells, which might be associated withpossible tumor cell seeding, immune responses, and xeno-zoonoses, areused. The availability of cells with fully mature hepatic function ofhuman origin, would enable investigators to further test and optimizeBAL devices to bridge patients till the liver spontaneously regeneratesor a donor-liver is available. Accordingly, a compound of the inventioncan be used to grow and differentiate hepatocytes for use withbioartificial liver devices.

Suspension-type bioartificial livers may comprise a compound of theinvention suspended with hepatocytes in plate dialysers,microencapsulated in a suitable substrate, or attached to microcarrierbeads coated with extracellular matrix. Alternatively, a compound of theinvention and hepatocytes can be placed on a solid support in a packedbed, in a multiplate flat bed, on a microchannel screen, or surroundinghollow fiber capillaries. The device has an inlet and outlet throughwhich the subject's blood is passed, and sometimes a separate set ofports for supplying nutrients to the cells.

Suspension-type bioartificial livers may comprise cells of the inventionsuspended in plate dialysers, microencapsulated in a suitable substrate,or attached to microcarrier beads coated with extracellular matrix.Alternatively, cells of the invention can be placed on a solid supportin a packed bed, in a multiplate flat bed, on a microchannel screen, orsurrounding hollow fiber capillaries. The device has an inlet and outletthrough which the subject's blood is passed, and sometimes a separateset of ports for supplying nutrients to the cells.

A compound of the invention and/or cells of the invention can be platedinto the device on a suitable substrate, such as a matrix of Matrigel®or collagen. The efficacy of the device can be assessed by comparing thecomposition of blood in the afferent channel with that in the efferentchannel—in terms of metabolites removed from the afferent flow, andnewly synthesized proteins in the efferent flow.

In certain embodiments, a compound of the invention may be used for thetreatment of viral hepatitis. In certain embodiments, a compound of theinvention and/or cells of the invention may be used to empower hepatitisresearch by providing for the development of novel disease models. Viralhepatitis is liver inflammation due to a viral infection. It may presentin acute (recent infection, relatively rapid onset) or chronic forms.The most common causes of viral hepatitis are the five unrelatedhepatotropic viruses Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D,and Hepatitis E. In addition to the hepatitis viruses, other virusesthat can also cause hepatitis include Herpes simplex, Cytomegalovirus,Epstein-Barr virus, or Yellow fever.

Organ and Tissue Transplant

A compound of the invention and/or cells of the invention may be used inmethods of organ or tissue transplantation. Organ transplantation is themoving of an organ from one body to another or from a donor site on thepatient's own body, for the purpose of replacing the recipient's damagedor absent organ. Organs may be re-grown from the patient's own cells(stem cells, or cells extracted from the failing organs). Organs and/ortissues that are transplanted within the same person's body are calledautografts. Transplants that are performed between two subjects of thesame species are called allografts. Allografts can either be from aliving or cadaveric source. Organs that can be transplanted includeliver.

The compounds of the invention and cells of the invention further enablethe development of implantable systems and/or tissue engineeredconstructs.

Cell Replacement Therapy

A compound of the invention and/or cells of the invention may be used ina method of cell replacement therapy. Cell replacement therapyintroduces new cells into damaged tissue in order to treat disease orinjury. The ability of cells to self-renew and give rise to subsequentgenerations with variable degrees of differentiation capacities allowsfor generating tissues that can replace diseased and damaged areas inthe body, with minimal risk of rejection and side effects.

Formulations & Administration

The compositions and methods of the present invention may be utilized totreat an individual in need thereof. In certain embodiments, theindividual is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered as a pharmaceutical compositioncomprising, for example, a compound of the invention and apharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known in the art andinclude, for example, aqueous solutions such as water or physiologicallybuffered saline or other solvents or vehicles such as glycols, glycerol,oils such as olive oil, or injectable organic esters. In a preferredembodiment, when such pharmaceutical compositions are for humanadministration, particularly for invasive routes of administration(i.e., routes, such as injection or implantation, that circumventtransport or diffusion through an epithelial barrier), the aqueoussolution is pyrogen-free, or substantially pyrogen-free. The excipientscan be chosen, for example, to effect delayed release of an agent or toselectively target one or more cells, tissues or organs. Thepharmaceutical composition can be in dosage unit form such as tablet,capsule (including sprinkle capsule and gelatin capsule), granule,lyophile for reconstitution, powder, solution, syrup, suppository,injection or the like. The composition can also be present in atransdermal delivery system, e.g., a skin patch. The composition canalso be present in a solution suitable for topical administration, suchas an eye drop.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as acompound of the invention. Such physiologically acceptable agentsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. The choice of a pharmaceutically acceptable carrier,including a physiologically acceptable agent, depends, for example, onthe route of administration of the composition. The preparation orpharmaceutical composition can be a selfemulsifying drug delivery systemor a selfmicroemulsifying drug delivery system. The pharmaceuticalcomposition (preparation) also can be a liposome or other polymermatrix, which can have incorporated therein, for example, a compound ofthe invention. Liposomes, for example, which comprise phospholipids orother lipids, are nontoxic, physiologically acceptable and metabolizablecarriers that are relatively simple to make and administer.

A pharmaceutical composition (preparation) can be administered to asubject by any of a number of routes of administration including, forexample, orally (for example, drenches as in aqueous or non-aqueoussolutions or suspensions, tablets, capsules (including sprinkle capsulesand gelatin capsules), boluses, powders, granules, pastes forapplication to the tongue); absorption through the oral mucosa (e.g.,sublingually); anally, rectally or vaginally (for example, as a pessary,cream or foam); parenterally (including intramuscularly, intravenously,subcutaneously or intrathecally as, for example, a sterile solution orsuspension); nasally; intraperitoneally; subcutaneously; transdermally(for example as a patch applied to the skin); and topically (forexample, as a cream, ointment or spray applied to the skin, or as an eyedrop). The compound may also be formulated for inhalation. In certainembodiments, a compound may be simply dissolved or suspended in sterilewater. Details of appropriate routes of administration and compositionssuitable for same can be found in, for example, U.S. Pat. Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any suitable methods. The amount of active ingredientwhich can be combined with a carrier material to produce a single dosageform will vary depending upon the host being treated, the particularmode of administration. The amount of active ingredient that can becombined with a carrier material to produce a single dosage form willgenerally be that amount of the compound which produces a therapeuticeffect. Generally, out of 100%, this amount will range from about 1% toabout 99% of active ingredient, preferably from about 5% to about 70%,most preferably from about 10% to about 30%.

Methods of preparing these formulations or compositions include the stepof bringing into association an active compound, such as a compound ofthe invention, with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules (including sprinkle capsules and gelatin capsules),cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), lyophile, powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a compound of the present invention as anactive ingredient. Compositions or compounds may also be administered asa bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules(including sprinkle capsules and gelatin capsules), tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; (10) complexing agents,such as, modified and unmodified cyclodextrins; and (11) coloringagents. In the case of capsules (including sprinkle capsules and gelatincapsules), tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragees, capsules (including sprinkle capsules andgelatin capsules), pills and granules, may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings well known in the pharmaceutical-formulating art. They may alsobe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms useful for oral administration includepharmaceutically acceptable emulsions, lyophiles for reconstitution,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, cyclodextrins and derivatives thereof, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.Pharmaceutical compositions suitable for parenteral administrationcomprise one or more active compounds in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

For use in the methods of this invention, active compounds can be givenper se or as a pharmaceutical composition containing, for example, 0.1to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount may include, but are not limited to, the severityof the patient's condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with the compound of the invention. A larger total dose canbe delivered by multiple administrations of the agent. Methods todetermine efficacy and dosage are known to those skilled in the art(Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the invention will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentinvention, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

In certain embodiments, compounds of the invention are administered atdosage levels greater than about 0.001 mg/kg, such as greater than about0.01 mg/kg or greater than about 0.1 mg/kg. For example, the dosagelevel may be from about 0.001 mg/kg to about 50 mg/kg such as from about0.01 mg/kg to about 25 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, orfrom about 1 mg/kg to about 5 mg/kg of subject body weight per day, oneor more times a day, to obtain the desired therapeutic effect. It willalso be appreciated that dosages smaller than 0.001 mg/kg or greaterthan 50 mg/kg (for example 50-100 mg/kg) can also be administered to asubject.

In certain embodiments, compounds of the invention may be used alone orconjointly administered with another type of therapeutic agent. As usedherein, the phrase “conjoint administration” refers to any form ofadministration of two or more different therapeutic compounds such thatthe second compound is administered while the previously administeredtherapeutic compound is still effective in the body (e.g., the twocompounds are simultaneously effective in the patient, which may includesynergistic effects of the two compounds). For example, the differenttherapeutic compounds can be administered either in the same formulationor in a separate formulation, either concomitantly or sequentially. Incertain embodiments, the different therapeutic compounds can beadministered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72hours, or a week of one another. Thus, an individual who receives suchtreatment can benefit from a combined effect of different therapeuticcompounds.

The compounds described herein may form salts with acids, and such saltsare included in the present invention. In one embodiment, the salts arepharmaceutically acceptable salts.

Toxicity and efficacy of the compounds of the invention can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds that exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

Data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the compounds of the inventionfor use in humans. The dosage of such agents lies preferably within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the effective dose can beestimated initially from cell culture assays. A dose may be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC₅₀ (i.e., the concentration of the test compoundthat achieves a half-maximal inhibition of symptoms) as determined incell culture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured, for example,by high performance liquid chromatography. In certain embodiments,pharmaceutical compositions may comprise, for example, at least about0.1% of an active compound. In other embodiments, the an active compoundmay comprise between about 2% to about 75% of the weight of the unit, orbetween about 25% to about 60%, for example, and any range derivabletherein. Multiple doses of the compounds of the invention are alsocontemplated.

Cell Administration

Any of the cells produced by the methods described herein can be used inthe clinic to treat a subject. They can, therefore, be formulated into apharmaceutical composition. Therefore, in certain embodiments, theisolated or purified cell populations are present within a compositionadapted for and suitable for delivery, i.e., physiologically compatible.Accordingly, compositions of the cell populations will often furthercomprise one or more buffers (e.g., neutral buffered saline or phosphatebuffered saline), carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. In certain embodiments, the isolated or purified cellpopulations are present within a composition adapted for or suitable forfreezing or storage.

Suitable means of delivery for administering the cells produced by themethods described herein include but are not limited to intravenousdelivery, direct delivery into the liver, delivery to other extrahepaticsites.

In certain embodiments, the purity of the cells for administration to asubject is about 100%. In certain embodiments, it is 95% to 100%. Incertain embodiments, it is 85% to 95%. Particularly in the case ofadmixtures with other cells, the percentage can be about 10%-15%,15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 60%-70%,70%-80%, 80%-90%, or 90%-95%. Or isolation/purity can be expressed interms of cell doublings where the cells have undergone, for example,10-20, 20-30, 30-40, 40-50 or more cell doublings.

The numbers of cells in a given volume can be determined by well knownand routine procedures and instrumentation. The % of the cells in agiven volume of a mixture of cells can be determined by much the sameprocedures. Cells can be readily counted manually or by using anautomatic cell counter. Specific cells can be determined in a givenvolume using specific staining and visual examination and by automatedmethods using specific binding reagent, typically antibodies,fluorescent tags, and a fluorescence activated cell sorter.

The choice of formulation for administering the cells for a givenapplication depends on a variety of factors. Prominent among these arethe species of subject, the nature of the disease or condition beingtreated and its state and distribution in the subject, the nature ofother therapies and agents that are being administered, the optimumroute for administration, survivability via the route, the dosingregimen, and other factors that will be apparent to those skilled in theart. In particular, for instance, the choice of suitable carriers andother additives will depend on the exact route of administration and thenature of the particular dosage form.

For example, cell survival can be an important determinant of theefficacy of cell-based therapies. This is true for both primary andadjunctive therapies. Another concern arises when target sites areinhospitable to cell seeding and cell growth. This may impede access tothe site and/or engraftment there of therapeutic cells. Variousembodiments of the invention comprise measures to increase cell survivaland/or to overcome problems posed by barriers to seeding and/or growth.

In certain embodiments, final formulations of the aqueous suspension ofcells/medium will typically involve adjusting the ionic strength of thesuspension to isotonicity (i.e., about 0.1 to 0.2) and to physiologicalpH (i.e., about pH 6.8 to 7.5). In certain embodiments, the finalformulation will also typically contain a fluid lubricant, such asmaltose, which must be tolerated by the body. Exemplary lubricantcomponents include glycerol, glycogen, maltose and the like. Organicpolymer base materials, such as polyethylene glycol and hyaluronic acidas well as non-fibrillar collagen, preferably succinylated collagen, canalso act as lubricants. Such lubricants are generally used to improvethe injectability, intrudability and dispersion of the injectedbiomaterial at the site of injection and to decrease the amount ofspiking by modifying the viscosity of the compositions. This finalformulation is by definition the cells in a pharmaceutically acceptablecarrier.

The cells are subsequently placed in a syringe or other injectionapparatus for precise placement at the site of the tissue defect. Theterm “injectable” means the formulation can be dispensed from syringeshaving a gauge as low as 25 under normal conditions under normalpressure without substantial spiking. Spiking can cause the compositionto ooze from the syringe rather than be injected into the tissue. Forthis precise placement, needles as fine as 27 gauge (200 μI.D.) or even30 gauge (150 μI.D.) are desirable. The maximum particle size that canbe extruded through such needles will be a complex function of at leastthe following: particle maximum dimension, particle aspect ratio(length:width), particle rigidity, surface roughness of particles andrelated factors affecting particle:particle adhesion, the viscoelasticproperties of the suspending fluid, and the rate of flow through theneedle. Rigid spherical beads suspended in a Newtonian fluid representthe simplest case, while fibrous or branched particles in a viscoelastic fluid are likely to be more complex.

The desired isotonicity of the cell compositions of this invention maybe accomplished using sodium chloride, or other pharmaceuticallyacceptable agents such as dextrose, boric acid, sodium tartrate,propylene glycol, or other inorganic or organic solutes. Sodium chlorideis preferred particularly for buffers containing sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the agent selected, andallows for the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative or stabilizer can be employedto increase the life of cell/medium compositions. If such preservativesare included, it is well within the purview of the skilled artisan toselect compositions that will not affect the viability or efficacy ofthe cells.

Those skilled in the art will recognize that the components of the cellcompositions should be chemically inert. This will present no problem tothose skilled in chemical and pharmaceutical principles. Problems can bereadily avoided by reference to standard texts or by simple experiments(not involving undue experimentation) using information provided by thedisclosure, the documents cited herein, and generally available in theart.

Sterile injectable solutions can be prepared by incorporating thecells/medium utilized in practicing the present invention in therequired amount of the appropriate solvent with various amounts of theother ingredients, as desired.

In some embodiments, the cells or medium are formulated in a unit dosageinjectable form, such as a solution, suspension, or emulsion.Pharmaceutical formulations suitable for injection of cells/mediumtypically are sterile aqueous solutions and dispersions. Carriers forinjectable formulations can be a solvent or dispersing mediumcontaining, for example, water, saline, phosphate buffered saline,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol, and the like), and suitable mixtures thereof.

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions to beadministered in methods of the invention. Typically, any additives (inaddition to the cells) are present in an amount of 0.001 to 50 wt % insolution, such as in phosphate buffered saline. The active ingredient ispresent in the order of micrograms to milligrams, such as about 0.0001to about 5 wt %, preferably about 0.0001 to about 1 wt %, mostpreferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt%, preferably about 0.01 to about 10 wt %, and most preferably about0.05 to about 5 wt %.

In some embodiments, cells are encapsulated for administration,particularly where encapsulation enhances the effectiveness of thetherapy, or provides advantages in handling and/or shelf life.Encapsulation in some embodiments where it increases the efficacy ofcell mediated immunosuppression may, as a result, also reduce the needfor immunosuppressive drug therapy.

Also, encapsulation in some embodiments provides a barrier to asubject's immune system that may further reduce a subject's immuneresponse to the cells (which generally are not immunogenic or are onlyweakly immunogenic in allogeneic transplants), thereby reducing anygraft rejection or inflammation that might occur upon administration ofthe cells. Cells may be encapsulated by membranes, as well as capsules,prior to implantation. Any of the many methods of cell encapsulationavailable may be employed. In some embodiments, cells are individuallyencapsulated. In some embodiments, many cells are encapsulated withinthe same membrane. In embodiments in which the cells are to be removedfollowing implantation, a relatively large size structure encapsulatingmany cells, such as within a single membrane, may provide a convenientmeans for retrieval.

A wide variety of materials may be used in various embodiments formicroencapsulation of cells. Such materials include, for example,polymer capsules, alginate-poly-L-lysine-alginate microcapsules, bariumpoly-L-lysine alginate capsules, barium alginate capsules,polyacrylonitrile/polyvinylchloride (PAN/PVC) hollow fibers, andpolyethersulfone (PES) hollow fibers.

Techniques for microencapsulation of cells that may be used foradministration of cells are known to those of skill in the art and aredescribed, for example, in European Patent Publication No. 301,777 andU.S. Pat. Nos. 5,639,275, 4,353,888; 4,744,933; 4,749,620; 4,814,274;5,084,350; 5,089,272; 5,578,442; 5,639,275; and 5,676,943. All of theforegoing is incorporated herein by reference in parts pertinent toencapsulation of cells.

Certain embodiments incorporate cells into a polymer, such as abiopolymer or synthetic polymer. Examples of biopolymers include, butare not limited to, fibronectin, fibin, fibrinogen, thrombin, collagen,and proteoglycans. Other factors, such as the cytokines discussed above,can also be incorporated into the polymer. In other embodiments of theinvention, cells may be incorporated in the interstices of athree-dimensional gel. A large polymer or gel, typically, will besurgically implanted. A polymer or gel that can be formulated in smallenough particles or fibers can be administered by other common, moreconvenient, non-surgical routes.

In the case of treating liver deficiency, in particular, the cells maybe enclosed in a device that can be implanted in a subject. Cells can beimplanted in or near the liver or elsewhere to replace or supplementliver function. Cells can also be implanted without being in a device,e.g., in existing liver tissue.

Cell compositions can be administered in dosages and by techniques wellknown to those skilled in the medical and veterinary arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular patient, and the formulation that will be administered (e.g.,solid vs. liquid). Doses for humans or other mammals can be determinedwithout undue experimentation by the skilled artisan, from thisdisclosure, the documents cited herein, and the knowledge in the art.

The dose of cells/medium appropriate to be used in accordance withvarious embodiments of the invention will depend on numerous factors. Itmay vary considerably for different circumstances. The parameters thatwill determine optimal doses to be administered for primary andadjunctive therapy generally will include some or all of the following:the disease being treated and its stage; the species of the subject,their health, gender, age, weight, and metabolic rate; the subject'simmunocompetence; other therapies being administered; and expectedpotential complications from the subject's history or genotype. Theparameters may also include: whether the cells are syngeneic,autologous, allogeneic, or xenogeneic; their potency (specificactivity); the site and/or distribution that must be targeted for thecells/medium to be effective; and such characteristics of the site suchas accessibility to cells/medium and/or engraftment of cells. Additionalparameters include co-administration with other factors (such as growthfactors and cytokines). The optimal dose in a given situation also willtake into consideration the way in which the cells/medium areformulated, the way they are administered, and the degree to which thecells/medium will be localized at the target sites followingadministration. Finally, the determination of optimal dosing necessarilywill provide an effective dose that is neither below the threshold ofmaximal beneficial effect nor above the threshold where the deleteriouseffects associated with the dose outweighs the advantages of theincreased dose.

In certain embodiments, for human therapy, the dose is generally betweenabout 10⁹ and 10¹² cells, making adjustments for body weight of thesubject, nature and severity of the affliction, and the replicativecapacity of the administered cells. In certain embodiments, the optimaldose of cells for some embodiments will be in the range of doses usedfor autologous, mononuclear bone marrow transplantation. For fairly purepreparations of cells, optimal doses in various embodiments will rangefrom 10⁴ to 10⁸ cells/kg of recipient mass per administration. In someembodiments the optimal dose per administration will be between 10⁵ to10⁷ cells/kg. In many embodiments the optimal dose per administrationwill be 5×10⁵ to 5×10⁶ cells/kg. By way of reference, higher doses inthe foregoing are analogous to the doses of nucleated cells used inautologous mononuclear bone marrow transplantation. Some of the lowerdoses are analogous to the number of CD34+ cells/kg used in autologousmononuclear bone marrow transplantation.

It is to be appreciated that a single dose may be delivered all at once,fractionally, or continuously over a period of time. The entire dosealso may be delivered to a single location or spread fractionally overseveral locations.

In various embodiments, cells/medium may be administered in an initialdose, and thereafter maintained by further administration. Cells/mediummay be administered by one method initially and thereafter administeredby the same method or one or more different methods. The levels can bemaintained by the ongoing administration of the cells/medium. Variousembodiments administer the cells/medium either initially or to maintaintheir level in the subject or both by intravenous injection. In avariety of embodiments, other forms of administration are used,dependent upon the patient's condition and other factors, discussedelsewhere herein.

It is noted that human subjects are generally treated longer thanexperimental animals; but, treatment generally has a length proportionalto the length of the disease process and the effectiveness of thetreatment. Those skilled in the art will take this into account in usingthe results of other procedures carried out in humans and/or in animals,such as rats, mice, non-human primates, and the like, to determineappropriate doses for humans. Such determinations, based on theseconsiderations and taking into account guidance provided by the presentdisclosure and the prior art will enable the skilled artisan to do sowithout undue experimentation.

Suitable regimens for initial administration and further doses or forsequential administrations may all be the same or may be variable.Appropriate regimens can be ascertained by the skilled artisan, fromthis disclosure, the documents cited herein, and the knowledge in theart.

The dose, frequency, and duration of treatment will depend on manyfactors, including the nature of the disease, the subject, and othertherapies that may be administered. Accordingly, a wide variety ofregimens may be used to administer the cells/medium.

In some embodiments, cells/medium are administered to a subject in onedose. In others cells/medium are administered to a subject in a seriesof two or more doses in succession. In some other embodiments whereincells/medium are administered in a single dose, in two doses, and/ormore than two doses, the doses may be the same or different, and theyare administered with equal or with unequal intervals between them.

Cells/medium may be administered in many frequencies over a wide rangeof times. In some embodiments, they are administered over a period ofless than one day. In other embodiment they are administered over two,three, four, five, or six days. In some embodiments they areadministered one or more times per week, over a period of weeks. Inother embodiments they are administered over a period of weeks for oneto several months. In various embodiments they may be administered overa period of months. In others they may be administered over a period ofone or more years. Generally lengths of treatment will be proportionalto the length of the disease process, the effectiveness of the therapiesbeing applied, and the condition and response of the subject beingtreated.

Kits

In various embodiments, the invention provides kits comprising acompound, a cell of the invention and/or a cell obtrainable using thecompounds or methods of the invention. Such kits are useful in in vitroand in vivo methods recited herein. In some embodiments, the kitprovides a sterile container comprising a composition of the invention;such containers can be boxes, ampoules, bottles, vials, tubes, bags,pouches, blister-packs, or other suitable container forms known in theart. Such containers can be made of plastic, glass, laminated paper,metal foil, or other materials suitable for holding reagents. If desireda composition of the invention is provided together with instructionsfor implementating a method of the invention. The instructions may beprinted directly on the container (when present), or as a label appliedto the container, or as a separate sheet, pamphlet, card, or foldersupplied in or with the container.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the compositions of the invention, and are not intended tolimit the scope of what the inventors regard as their invention.

EXAMPLES Methods & Materials

The compounds of the invention can be synthesized according to methodsknown in the art, and/or as outlined in Schemes 1 and 2 providedelsewhere herein.

Starting materials 1 and 2 may be prepared according to Marcaurelle, etal., 2010, J. Am. Chem. Soc. 132:16962-16976.

Compounds were purchased from commercial suppliers and analyzed byliquid chromatography (FIGS. 14A, 15A and 16), ¹H-NMR mass spectrometry(FIGS. 14B and 15B) and ¹³C-NMR mass spectrometry (FIGS. 14C and 15C).FPH1 and FH1 were purchased as a dry powder from Molport. FPH2 waspurchased as a dry powder from ChemBridge Corporation.

FPH1:

LCMS: Expected [M+H]+ 354.0786, Determined [M+H]+ 354.0793; HPLC: 94% (@210 nm) (Rt; 0.60). ¹H NMR (300 MHz, DMSO-_(d6)) δ 10.32 (s, 1H), 9.99(s, 1H), 8.85 (s, 1H), 7.63 (s, 1H), 7.46 (s, 2H), 7.31 (dd, J=8.8, 2.4Hz, 1H), 7.12 (d, J=8.9 Hz, 1H), 4.17 (q, J=7.2 Hz, 2H), 3.77 (s, 3H),3.34 (s, 2H), 1.37 (t, J=7.2 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ177.14, 165.26, 152.74, 132.23, 127.58, 127.29, 127.11, 124.73, 123.52,121.47, 113.76, 55.86, 47.10, 15.26.

FPH2:

LCMS: Expected [M+H]+ 389.0533, Determined [M+H]+ 389.0534; HPLC: 98% (@210 nm) (Rt; 0.66). ¹H NMR (300 MHz, DMSO-d₆) δ 9.93 (s, 1H), 7.61 (s,1H), 7.36 (m, 3H), 7.16 (t, J=8.2 Hz, 2H), 3.34 (s, 2H), 3.21 (s, 3H),2.33 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ 167.15, 159.13, 139.97,138.04, 132.50, 130.28, 129.30, 128.62, 128.29, 113.66, 112.04, 111.73,52.95, 17.50.

Luminex Analysis

Cells were lysed using RLT buffer (Qiagen) or Trizol (Invitrogen) andpurified using the Mini-RNeasy kit (Qiagen). Gene expression wasdetermined using Luminex analysis, as previously described. Briefly,total RNA was immobilized on a Qiagen turbo capture 384-well plate, andreverse-transcribed using oligodT priming A biotinylated FlexMap tagsequence unique to each gene of interest and a phosphorylated downstreamprobe were then added to resulting cDNAs to generate biotinylatedFlexMap-tagged amplicons. Universal PCR was then performed for 35 cyclesusing a biotinylated T7 forward primer and T3 reverse primer in bufferwith dNTPs and Taq polymerase. FlexMap microsphere beads conjugated withantitag oligonucleotides were then added and allowed to hybridize.Amplicons were captured by streptavidin-phyoerythrin, and 100 events perbead were analyzed for internal bead color and phyoerythrin reporterfluorescence on a Luminex FlexMap 3D analyzer. Data for replicateloadings, expressed in mean fluorescent intensity of at least 100 beadsper sample, were scaled to the human transferrin gene and row-normalizedfor heat map representation using GeneE open software (Broad Institute).

Biochemical Assays

Culture media were collected and frozen at −20° C. until analysis.Albumin content was measured through sandwich ELISA assays (MPBiomedicals, Fitzgerald, Bethyl Laboratories) using horseradishperoxidase detection and 3,3′,5,5′-tetramethylbenzidine (TMB, FitzgeraldIndustries) as a substrate. Urea concentration was determinedcolorimetrically using diacetylmonoxime with acid and heat (StanbioLabs). To quantify CYP450 activity, at 48 hours after small moleculeexposure, cultures were incubated with substrates (coumarin from Sigmafor CYP2A6, luciferin-IPA from Promega for CYP3A4) for 4 hours at 37° C.Incubation medium was collected and metabolite concentration quantifiedvia luminescence, or fluorescence after hydrolization of potentialmetabolite conjugates by β-glucuronidase/arylsulfatase (Roche, Ind.).

Example 1: Preparation of Compounds 2 and 4 (Scheme 1)

General Protocol for Coupling of TBS-Protected Acid with PMB-ProtectedAlaninol

tert-Butyl2-(tert-butyldimethylsilyloxy)-4-(1-(4-methoxybenzyloxy)propan-2-ylamino)-3-methyl-4-oxobutyl(methyl)carbamate(13)

An oven-dried, 3-L, 3-neck round bottom flask was equipped with anoverhead stirrer, addition funnel and a temperature probe. Under apositive flow of N₂, the vessel was charged with4-(tert-butoxycarbonyl(methyl)amino)-3-(tert-butyldimethylsilyloxy)-2-methylbutanoicacid 1 (1.0 equiv) dissolved in CH₂Cl₂ (80% of total solvent, finalconcentration of 1 was 0.2 M), followed by PyBOP (1.0 equiv), anddiisopropyl ethylamine (DIPEA) (3.0 equiv). The resulting mixture wascooled in an ice bath before 1-(4-methoxybenzyloxy)propan-2-amine 2(1.1-1.2 equiv) was added as a solution in CH₂Cl₂ (remaining 20% oftotal solvent) by addition funnel. The rate of addition was controlledso as to maintain an internal temperature between 3-5° C. When additionwas complete, the mixture was warmed to ambient temperature and allowedto stir for 15 h. The reaction was quenched with water, and extractedwith CH₂Cl₂. The combined organic extracts were dried over MgSO₄,filtered and concentrated. The yellow oil was taken up in ethyl ether,and the phosphoramide byproducts were removed via filtration. Thesolvent was removed in vacuo and the crude product was isolated. Flashchromatography on silica gel (4:1 Hexanes/EtOAc to 7:3 Hexanes/EtOAc)gave the product 13 as a colorless oil.

Linear Amide (13a)

Following the general reaction protocol (−)-1a (124 g, 343 mmol, 1.0equiv) was reacted with PyBOP (179 g, 343 mmol, 1.0 equiv), DIPEA (180mL, 1030 mmol, 3.0 equiv) and (R)-alaninol (−)-2 (73.7 g, 378 mmol, 1.1equiv) in CH₂Cl₂ (1450 mL), which provided pure product (2R,5R,6R)-13(176 g, 95%) as a clear oil.

(2R,5R,6R)-13a: [α]_(D) ²⁰ −22.0 (c 1.0, CHCl₃). IR (cm⁻¹) 3337, 2930,1695, 1673, 1513, 1460, 1390, 1364, 1247, 1156. ¹H NMR (500 MHz, CDCl₃,55° C.) δ 7.20 (d, J=8.5 Hz, 2H), 6.84 (d, J=8.5 Hz, 2H), 6.57 (br s,1H, NH), 4.46 (d, J=11.6, 1H), 4.40 (d, J=11.6, 1H), 4.24-4.17 (m, 1H),4.12-4.05 (m, 1H), 3.77 (s, 3H), 3.46 (dd, J=3.7, 14.4 Hz, 1H), 3.43(dd, J=4.4, 9.6 Hz, 1H), 3.38 (dd, J=4.8, 9.6 Hz, 1H), 3.03-2.87 (m,1H), 2.79 (s, 3H), 2.38 (dq, J=3.7, 7.2 Hz, 1H), 1.44 (s, 9H), 1.15 (d,J=6.7 Hz, 3H), 1.12 (d, J=7.2, 3H), 0.90 (s, 9H), 0.07 (s, 3H), 0.05 (s,3H). ¹³C NMR (125 MHz, CDCl₃, 55° C.) δ 172.4 (br), 159.4, 155.7 (br),130.4, 129.1 (2C), 113.8 (2C), 79.4 (br), 73.9 (br), 72.9, 72.8, 55.2,51.5, 44.7, 44.5, 36.7 (br), 28.5 (3C), 25.8 (3C), 17.8, 17.7, 12.4,−4.8, −4.9. HRMS (ESI) calcd for C₂₈H₅₀N₂NaO₉Si [M+Na]⁺: 561.3330.Found: 561.3351.

(2S,5S,6S)-13a: [α]_(D) ²⁰ +26.2 (c 1.0, CHCl₃).

Linear Amide (13b)

Following the general reaction protocol, (−)-1a (131 g, 362 mmol, 1.0equiv) was reacted with PyBOP (189 g, 362 mmol, 1.0 equiv), DIPEA (190mL, 1087 mmol, 3.0 equiv) and (S)-alaninol (+)-2 (78 g, 399 mmol, 1.1equiv) in CH₂Cl₂ (1812 mL), which provided pure product (2S,5R,6R)-13b(156 g, 80%) as a clear oil.

(2S,5R,6R)-13b: [α]_(D) ²⁰ −39.9 (c 1.0, CHCl₃). IR (cm⁻¹) 3338, 2930,1697, 1675, 1513, 1460, 1390, 1365, 1248, 1157. ¹H NMR (500 MHz, CDCl₃,55° C.) δ 7.21 (d, J=8.5 Hz, 2H), 6.85 (d, J=8.5 Hz, 2H), 6.41 (br s,1H, NH), 4.45 (d, J=11.7, 1H), 4.41 (d, J=11.7, 1H), 4.20-4.14 (m, 1H),4.13-4.04 (m, 1H), 3.78 (s, 3H), 3.47 (dd, J=3.6, 14.3 Hz, 1H), 3.39 (d,J=4.3 Hz, 2H), 3.00-2.85 (m, 1H), 2.86 (s, 3H), 2.38 (dq, J=3.8, 7.2 Hz,1H), 1.45 (s, 9H), 1.19 (d, J=6.7 Hz, 3H), 1.12 (d, J=7.2, 3H), 0.89 (s,9H), 0.07 (s, 3H), 0.05 (s, 3H). ¹³C NMR (125 MHz, CDCl₃, 55° C.) δ172.3 (br), 159.4, 155.8 (br), 130.5, 129.2 (2C), 113.9 (2C), 79.5 (br),73.5 (br), 72.93, 72.85, 55.2, 51.8, 44.87, 44.85, 36.7 (br), 28.5 (3C),25.9 (3C), 17.89, 17.88, 12.6, −4.7, −4.8. HRMS (ESI) calcd forC₂₈H₅₀N₂NaO₆Si [M+Na]⁺: 561.3330. Found: 561.3313.

(2R,5S,6S)-13b: [α]_(D) ²⁰ +42.1 (c 1.0, CHCl₃).

Linear Amide (13c)

Following the general reaction protocol, (−)-1b (38.1 g, 105 mmol, 1.0equiv) was reacted with PyBOP (60.4 g, 116 mmol, 1.1 equiv), DIPEA (55.3mL, 316 mmol, 3.0 equiv) and (R)-alaninol (−)-2 (24.7 g, 127 mmol, 1.2equiv) in CH₂Cl₂ (820 mL), which provided pure product (2R,5R,6S)-13c(51.9 g, 91%) as a clear oil.

(2R,5R,6S)-13c: [α]_(D) ²⁰ +17.6 (c 1.0, CHCl₃). IR (cm⁻¹) 3365, 2930,1696, 1670, 1513, 1460, 1390, 1365, 1248, 1156. ¹H NMR (500 MHz, CDCl₃,55° C.) δ 7.22 (d, J=8.5 Hz, 2H), 6.86 (d, J=8.5 Hz, 2H), 6.75 (br s,1H, NH), 4.46 (d, J=11.7, 1H), 4.41 (d, J=11.7, 1H), 4.22-4.15 (m, 1H),4.05-3.98 (m, 1H), 3.80 (s, 3H), 3.39 (d, J=4.6 Hz, 2H), 3.34 (dd,J=6.4, 14.0 Hz, 1H), 3.18 (dd, J=6.3, 14.0 Hz, 1H), 2.88 (s, 3H), 2.41(dq, J=2.9, 7.3 Hz, 1H), 1.46 (s, 9H), 1.22 (d, J=7.2 Hz, 3H), 1.18 (d,J=6.7 Hz, 3H), 0.91 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H). ¹³C NMR (125MHz, CDCl₃, 55° C.) δ 173.4, 159.4, 156.0 (br), 130.6, 129.1 (2C), 113.9(2C), 79.7 (br), 72.9, 72.8, 72.6 (br), 55.3, 53.5, 44.8, 44.5, 36.6(br), 28.5 (3C), 25.9 (3C), 18.0, 17.9, 15.6, −4.5, −4.9. HRMS (ESI)calcd for C₂₈H₅₀N₂NaO₆Si [M+Na]⁺: 561.3330. Found: 561.3498.

(2S,5S,6R)-13c: [<]_(D) ²⁰ −16.5 (c 1.0, CHCl₃).

Linear Amide (13d)

Following the general reaction protocol, (−)-1b (92 g, 254 mmol, 1.0equiv) was reacted with PyBOP (132 g, 254 mmol, 1.0 equiv), DIPEA (133mL, 763 mmol, 3.0 equiv) and (S)-alaninol (+)-2 (59.6 g, 305 mmol, 1.2equiv) in CH₂Cl₂ (1957 mL), which provided pure product (S,R,S)-13d (145g, 70%) as a clear oil.

(2S,5R,6S)-13d: [α]_(D) ²⁰ −0.6 (c 1.0, CHCl₃). IR (cm⁻¹) 3356, 2930,1695, 1669, 1514, 1466, 1390, 1365, 1248, 1156. ¹H NMR (500 MHz, CDCl₃,55° C.) δ 7.20 (d, J=8.5 Hz, 2H), 6.87 (br s, 1H, NH), 6.85 (d, J=8.5Hz, 2H), 4.46 (d, J=11.6, 1H), 4.42 (d, J=11.6, 1H), 4.26-4.17 (m, 1H),4.02-3.96 (m, 1H), 3.78 (s, 3H), 3.44 (dd, J=4.4, 9.5 Hz, 1H), 3.40-3.30(m, 1H), 3.36 (dd, J=5.0, 9.5 Hz, 1H), 3.18-3.11 (m, 1H), 2.80 (s, 3H),2.39 (dq, J=2.8, 7.2 Hz, 1H), 1.44 (s, 9H), 1.22 (d, J=7.3 Hz, 3H), 1.14(d, J=6.7, 3H), 0.92 (s, 9H), 0.11 (s, 6H). ¹³C NMR (125 MHz, CDCl₃, 55°C.) δ 173.6, 159.4, 155.9 (br), 130.5, 129.2 (2C), 113.9 (2C), 79.6(br), 73.7 (br), 73.0, 72.8, 55.3, 53.5, 44.6, 44.4, 36.8, 28.5 (3C),25.9 (3C), 18.0, 17.7, 16.0, −4.3, −4.8. HRMS (ESI) calcd forC₂₈H₅₀N₂NaO₆Si [M+Na]⁺: 561.3330. Found: 561.3331.

(2R,5S,6R)-13d: [α]_(D) ²⁰ +1.4 (c 1.0, CHCl₃).

General Protocol for the Reduction of Amide

tert-Butyl2-(tert-butyldimethylsilyloxy)-4-(1-(4-methoxybenzyloxy)propan-2-ylamino)-3-methylbutyl(methyl)carbamate(3)

An oven-dried, 2-L, 1-necked round bottom flask was equipped with amagnetic stirrer. Under a positive flow of N₂, the flask was chargedwith tert-butyl2-(tert-butyldimethylsilyloxy)-4-(1-(4-methoxybenzyloxy)propan-2-ylamino)-3-methyl-4-oxobutyl(methyl)carbamate13 (1.0 equiv) and anhydrous THF (final concentration 0.1 M). Boranedimethylsulfide complex (BH₃DMS) (5.0 equiv) was added dropwise viasyringe. Afterwards the reaction mixture was heated at 65° C. for 5 h.After cooling to ambient temperature, excess hydride was quenched by thecareful addition of MeOH. The mixture was concentrated under reducedpressure to afford a colorless oil, which was then co-evaporated withMeOH three times to remove excess B(OMe)₃. The oil was then re-dissolvedin MeOH and 10% aqueous potassium sodium tartrate (2:3 ratio, finalconcentration 0.067 M). The resulting slurry was heated at reflux for 12h. The volatiles were removed under reduced pressure and aqueous layerwas extracted three times with ethyl acetate. The combined organiclayers were washed once with brine, dried over magnesium sulfate,filtered and concentrated to provide the desired amine 3 as a colorlessoil.

Linear Amine (3a)

Following the general reaction protocol (2R,5R,6R)-13a (100 g, 186 mmol,1.0 equiv) was reacted with BH₃DMS (88.0 mL, 930 mmol, 5.0 equiv) in THF(1860 mL), and worked up with 10% aqueous potassium sodium tartrate (2L) in MeOH (1.70 L), which provided pure product (2R,5S,6R)-(−)-3a (92.7g, 95%) as a clear oil.

(2R,5S,6R)-(−)-3a: [α]_(D) ²⁰ −16.15 (c 1.38, MeOH). IR (cm⁻¹) 3010,2929, 1687, 1513, 1462, 1389, 1247, 1153. ¹H NMR (500 MHz, CDCl₃, 60°C.) δ 7.20 (d, J=8.5 Hz, 2H), 6.83 (d, J=8.5 Hz, 2H), 4.41 (s, 2H), 3.95(ddd, J=11, 6, 5 Hz, 1H), 3.76 (s, 3H), 3.36 (dd, J=14, 6 Hz, 1H), 3.31(m, 2H), 2.97 (dd, J=14, 7 Hz, 1H), 2.83 (m, 4H), 2.60 (dd, J=11, 6 Hz,1H), 2.45 (dd, J=11, 6 Hz, 1H), 1.65 (ddd, J=12, 6, 5 Hz, 1H), 1.43 (s,9H), 0.98 (d, J=6 Hz, 3H), 0.90 (d, J=6 Hz, 3H), 0.87 (s, 9H), 0.03 (s,6H). ¹³C NMR (125 MHz, CDCl₃, 60° C.) δ 159.4, 155.7, 130.8, 129.1,113.9, 79.3, 74.7, 72.9, 72.3, 55.2, 52.9, 52.3, 50.1, 37.3, 36.2, 28.6,26.0, 18.1, 17.5, 12.8, −4.7. HRMS (ESI) calcd for C₂₈H₅₃N₂O₅Si [M+H]⁺:525.3718. Found: 525.3709.

(2S,5R,6S)-(+)-3a: [α]_(D) ²⁰ +19.09 (c 2.98, CHCl₃).

Linear Amine (3b)

Following the general reaction protocol, (2S,5R,6R)-13b (33.15 g, 61.5mmol, 1.0 equiv) was reacted with BH₃DMS (29.2 mL, 308 mmol, 5.0 equiv)in THF (615 mL), and worked up with 10% aqueous potassium sodiumtartrate (650 mL) in MeOH (450 mL), which provided pure product(2S,5S,6R)-(−)-3b (30.96 g, 96%) as a clear oil.

(2S,5S,6R)-(−)-3b: [α]_(D) ²⁰ −13.82 (c 1.23, MeOH). IR (cm⁻¹) 3010,2929, 1692, 1513, 1461, 1389, 1247, 1152. ¹H NMR (500 MHz, CDCl₃, 60°C.) δ 7.14 (d, J=8.5 Hz, 2H), 6.76 (d, J=8.5 Hz, 2H), 4.36 (d, J=12 Hz,1H), 4.33 (d, J=12 Hz, 1H), 3.94 (ddd, J=11, 6, 5 Hz, 1H), 3.68 (s, 3H),3.32-3.26 (m, 2H), 3.22 (dd, J=14, 7 Hz, 1H), 2.91 (dd, J=13, 6.5 Hz,1H), 2.76 (m, 4H), 2.61 (m, 1H), 2.31 (dd, J=11, 7.5 Hz, 1H), 1.60 (m,1H), 1.38 (s, 9H), 0.93 (d, J=6 Hz, 3H), 0.83 (d, J=6 Hz, 3H), 0.82 (s,9H), −0.02 (s, 3H), −0.028 (s, 3H). ¹³C NMR (125 MHz, CDCl₃, 60° C.) δ159.3, 155.7, 130.7, 129.1, 113.9, 79.2, 74.6, 72.9, 71.7, 55.1, 52.9,52.3, 50.1, 36.9, 36.0, 28.5, 26.0, 18.0, 17.6, 12.6, −4.6. HRMS (ESI)calcd for C₂₈H₅₃N₂O₅Si [M+H]⁺: 525.3718. Found: 525.3702.

(2R,5R,6S)-(+)-3b: [α]_(D) ²⁰ +15.89 (c 7.30, CHCl₃).

Linear Amine (3c)

Following the general reaction protocol, (2R,5R,6S)-13c (36.0 g, 66.8mmol, 1.0 equiv) was reacted with BH₃DMS (31.7 mL, 334 mmol, 5.0 equiv)in THF (668 mL), and worked up with 10% aqueous potassium sodiumtartrate (900 mL) in MeOH (600 mL), which provided pure product(2R,5S,6S)-(+)-3c (31.5 g, 90%) as a clear oil.

(2R,5S,6S)-(+)-3c: [α]_(D) ²⁰ +7.77 (c 0.99, MeOH). IR (cm⁻¹) 3010,2929, 1693 1513 1461, 1389, 1247, 1152. ¹H NMR (500 MHz, CDCl₃, 60° C.)δ 7.19 (d, J=8.5 Hz, 2H), 6.82 (d, J=8.5 Hz, 2H), 4.40 (s, 2H), 3.89 (m,1H), 3.74 (s, 3H), 3.29 (m, 4H), 2.98 (dd, J=13.5, 7.5 Hz, 1H), 2.85 (m,4H), 2.63 (m, 1H), 2.41 (dd, J=11, 9 Hz, 1H), 1.72 (m, 1H), 1.41 (s,9H), 0.98 (d, J=6.5 Hz, 1H), 0.92 (d, J=6.5 Hz, 1H), 0.86 (s, 9H), 0.02(s, 3H), 0.00 (s, 3H). ¹³C NMR (125 MHz, CDCl₃, 60° C.) δ 159.3, 155.7,130.8, 129.1, 113.9, 79.1, 74.6, 73.3, 72.8, 55.2, 52.5, 52.0, 49.4,38.7, 36.4, 28.6, 26.0, 18.0, 17.6, 13.7, −4.5. HRMS (ESI) calcd forC₂₈H₅₃N₂O₅Si [M+H]⁺: 525.3718. Found: 525.3698.

(2S,5R,6R)-(−)-3c: [α]_(D) ²⁰ −10.65 (c 2.46, CHCl₃).

Linear Amine (3d)

Following the general reaction protocol, (2S,5R,6S)-13d (55.0 g, 102mmol, 1.0 equiv) was reacted with BH₃DMS (48.4 mL, 510 mmol, 5.0 equiv)in THF (1030 mL), and worked up with 10% aqueous potassium sodiumtartrate (900 mL) in MeOH (600 mL), which provided pure product(2S,5S,6S)-(−)-3d (52.76 g, 98%) as a clear oil.

(2S,5S,6S)-(+)-3d: [α]_(D) ²⁰ +13.7 (c 2.62, CHCl₃). IR (cm⁻¹) 3010,2929, 1692, 1512, 1461, 1389, 1247, 1152. ¹H NMR (500 MHz, CDCl₃, 60°C.) δ 7.19 (d, J=8.5 Hz, 2H), 6.81 (d, J=8.5 Hz, 2H), 4.41 (d, J=12 Hz,1H), 4.39 (d, J=12 Hz, 1H), 3.92-3.90 (m, 1H), 3.74 (s, 3H), 3.35-3.28(m, 3H), 2.98 (dd, J=14, 7.5 Hz, 1H), 2.84-2.80 (m, 4H), 2.70 (m, 1H),2.32 (dd, J=11, 7.5 Hz, 1H), 1.75 (ddd, J=14, 7 Hz, 1H), 1.42 (s, 9H),0.98 (d, J=6.5 Hz, 3H), 0.91 (d, J=6.5 Hz, 3H), 0.86 (s, 9H), 0.02 (s,3H), 0.00 (s, 3H). ¹³C NMR (125 MHz, CDCl₃, 60° C.) δ 159.4, 155.9,130.8, 129.2, 114.0, 79.3, 74.6, 73.3, 73.0, 55.3, 53.3, 52.0, 49.8,38.7, 36.5, 28.7, 26.1, 18.1, 17.6, 13.7, −4.2. HRMS (ESI) calcd forC₂₈H₅₃N₂O₅Si [M+H]⁺: 525.3718. Found: 525.3718.

(2R,5R,6R)-(−)-3d: [α]_(D) ²⁰ −15.07 (c 6.40, CHCl₃).

General Protocol for Preparation of S_(N)Ar Precursors (S_(N)Ar-Pyr)

Triethylamine (4.0 equiv) was added to a solution of amine 3 (1.0 equiv)and 5-bromo-2-chloronicotinoyl chloride (Cho, et al., 2010, J. Bioorg.Med. Chem. Lett. 20:4223-4227) 14 (2.0 equiv) in CH₂Cl₂ (0.2 M) at 0° C.under dry nitrogen atmosphere. The reaction was warmed to RT and stirreduntil complete consumption of starting amine was observed (1-2 h). Thereaction was quenched with a saturated NH₄Cl solution and the resultingmixture was extracted with CH₂Cl₂. The combined organic extracts weredried over MgSO₄, filtered and concentrated. Flash chromatography onsilica gel (gradient: 10% to 30% EtOAc in hexanes) provided the product9 as a white foaming solid.

S_(N)Ar Precursor (9a)

Following the general reaction protocol (2R,5S,6R)-3a (26.4 g, 50.3mmol, 1.0 equiv) was reacted with 5-bromo-2-chloronicotinoyl chloride 14(25.6 g, 101 mmol, 2.0 equiv) and triethylamine (27.9 mL, 201 mmol, 4.0equiv) in CH₂Cl₂ (250 mL), which provided pure product (2R,5S,6R)-9a(35.6 g, 95%).

(2R,5S,6R)-9a: [α]_(D) ²⁰ +9.1 (c 1.0, CHCl₃). IR (cm⁻¹) 2954, 2930,2857, 1696, 1645, 1249, 1155, 836. ¹H NMR (500 MHz, CDCl₃, 2.3:1 mixtureof rotamers) δ 8.47-8.42 (m, 1H, ×0.3), 8.39-8.35 (m, 1H×0.7), 7.73-7.69(m, 1H×0.3), 7.57-7.53 (m, 1H×0.7), 7.24 (d, J=8.5 Hz, 2H×0.3), 7.19 (d,J=8.5 Hz, 2H×0.7), 6.90-6.83 (m, 2H), 4.50-4.41 (m, 2H×0.3), 4.39-4.31(m, 2H×0.7), 4.01-3.85 (m, 1H), 3.80 (s, 3H×0.7), 3.78 (s, 3H×0.3),3.85-3.56 (m, 2H), 3.51-3.32 (m, 1H), 3.27-3.09 (m, 1H), 3.20 (d, J=10.0Hz, 1H), 2.90 (d, J=13.6 Hz, 2H), 2.75 (d, J=10.5 Hz, 1H), 2.12 (br s,1H×0.7), 1.93 (br s, 1H×0.3), 1.45 (s, 9H), 1.40 (d, J=6.9 Hz, 3H×0.7),1.17 (d, J=6.9 Hz, 3H×0.3), 1.10 (s, 9H×0.3), 0.92 (s, 9H×0.7), 0.73 (s,3H×0.3), 0.68 (s, 3H×0.7), 0.07 (s, 3H×0.7), 0.03 (s, 3H×0.3), 0.06 (s,3H×0.7), −0.14 (s, 3H×0.3). ¹³C NMR (125 MHz, CDCl₃, reported as amixture of rotamers) δ 167.5, 165.9, 159.7, 159.4, 156.1, 155.8, 150.7,150.4, 145.6, 145.3, 141.9, 140.2, 138.9, 138.5, 135.0, 134.9, 129.9,129.8, 129.7, 129.6, 119.5, 119.3, 114.3, 114.1, 114.0, 113.9, 79.9,79.6, 74.1, 73.3, 73.2, 73.1, 70.3, 70.2, 55.5, 55.4, 55.1, 55.0, 52.0,51.9, 43.1, 42.7, 38.5, 38.0, 36.8, 36.7, 28.8, 28.7, 26.3, 26.2, 18.2,17.9, 16.5, 16.4, 13.3, 13.0, −4.2. HRMS (ESI) calcd forC₃₄H₅₅BrClN₃O₆Si [M+H]⁺: 742.2576. Found: 742.2642.

S_(N)Ar Precursor (9b)

Following the general reaction protocol (2S,5S,6R)-3b (26.2 g, 49.9mmol, 1.0 equiv) was reacted with 5-bromo-2-chloronicotinoyl chloride 14(25.5 g, 100 mmol, 2.0 equiv) and triethylamine (27.7 mL, 200 mmol, 4.0equiv) in CH₂Cl₂ (250 mL), which provided pure product (2S,5S,6R)-9b(34.8 g, 94%).

(2S,5S,6R)-9b: [α]_(D) ²⁰ −36.2 (c 1.8, CHCl₃). IR (cm⁻¹) 2954, 2930,2857, 1687, 1637, 1513, 1462, 1401, 1249, 1155, 1098, 1034. ¹H NMR (500MHz, CDCl₃, 3:1 mixture of rotamers) δ 8.38 (s, 1H×0.25), 8.33 (s,1H×0.75), 7.69 (s, 1H×0.25), 7.59 (s, 1H×0.75), 7.24-7.16 (2H, m),6.90-6.81 (m, 2H×0.75), 6.89-6.80 (m, 2H×0.25), 4.50-4.40 (m, 2H x0.25), 4.34-4.26 (m, 2H×0.75), 4.01-3.96 (m, 1H×0.25), 3.89-3.83 (m,1H×0.75), 3.77 (s, 3H×0.75), 3.75 (s, 3H×0.25), 3.72-3.65 (m, 1H),3.60-3.55 (m, 1H), 3.35-3.24 (m, 3H), 3.00 (s, 3H), 2.86-2.81 (m, 1H),2.47-2.42 (m, 1H), 1.47 (s, 9H), 1.19 (s, 3H×0.75) 1.05 (s, 3H×0.25),0.92 (s, 9H), 0.90-0.70 (m, 3H), 0.11-0.09 (m, 6H). ¹³C NMR (125 MHz,CDCl₃, reported as a mixture of rotamers)

167.5, 167.4, 159.7, 155.9, 150.5, 150.4, 145.1, 134.8, 134.7, 129.8,129.6, 119.4, 114.3, 113.9, 79.7, 79.4, 74.0, 73.3, 73.2, 73.0, 71.0,55.5, 54.9, 54.8, 52.4, 51.9, 43.4, 43.0, 36.8, 36.6, 35.7, 35.6, 35.0,28.9, 28.8, 28.6, 26.2, 25.9, 18.2, 18.0, 16.0, 15.9, 14.4, −4.3. HRMS(ESI) calcd for C₃₄H₅₅BrClN₃O₆Si [M+H]⁺: 742.2576. Found: 742.2662.

S_(N)Ar Precursor (9c)

Following the general reaction protocol, (2R,5S,6S)-3c (36.0 g, 68.6mmol, 1.0 equiv) was reacted with 5-bromo-2-chloronicotinoyl chloride 14(35.0 g, 137 mmol, 2.0 equiv) and triethylamine (38.0 mL, 274 mmol, 4.0equiv) in CH₂Cl₂ (500 mL), which provided pure product (2R,5S,6S)-9c(42.8 g, 84%).

(2R,5S,6S)-9c: [α]_(D) ²⁰ +19.9 (c 1.0, CHCl₃). IR (cm⁻¹) 2956, 2933,2857, 1693, 1643, 1249, 1157, 837. ¹H NMR (500 MHz, CDCl₃, 2:1 mixtureof rotamers) δ 8.46 (br s, 1H×0.33), 8.37 (br s, 1H×0.66), 7.69 (br s,1H×0.33), 7.61 (br s, 1H×0.66), 7.24 (d, J=7.5 Hz, 2H×0.33), 7.18 (d,J=7.5 Hz, 2H×0.66), 6.91-6.85 (m, 2H), 4.50-4.22 (m, 2H), 4.16-4.10 (m,1H×0.33), 3.97-3.91 (m, 1H×0.66), 3.81 (s, 3H×0.66), 3.79 (s, 3H×0.33),3.69-3.62 (m, 1H), 3.57-3.51 (m, 1H), 3.47-3.29 (m, 1H), 3.29-3.16 (m,1H), 3.13-2.98 (m, 1H), 2.96-2.89 (m, 2H), 2.84-2.76 (m, 1H), 2.13 (brs, 1H×0.66), 1.92 (br s, 1H×0.33), 1.46 (s, 9H×0.66), 1.44 (s, 9H×0.33),1.43 (s, 3H×0.33), 1.18 (s, 3H×0.66), 1.10 (s, 9H×0.33), 0.92 (s,9H×0.66), 0.75 (s, 3H×0.33), 0.66 (s, 3H×0.66), 0.08 (s, 3H×0.66), −0.05(s, 3H×0.33), −0.06 (s, 3H×0.66), −0.13 (s, 3H×0.33). ¹³C NMR (125 MHz,CDCl₃, reported as a mixture of rotamers) δ 167.2, 165.9, 159.6, 159.2,156.1, 155.9, 150.4, 150.2, 141.9, 141.7, 140.0, 138.5, 135.1, 134.8,129.7, 129.5, 129.4, 129.3, 119.3, 119.2, 114.2, 114.0, 79.8, 79.5,74.4, 73.3, 73.1, 72.9, 71.8, 70.4, 55.5, 55.4, 55.1, 54.9, 52.6, 52.0,51.8, 50.3, 42.8, 42.2, 38.4, 38.1, 37.3, 36.8, 28.7, 28.5, 26.1, 25.8,18.1, 17.9, 16.3, 15.9, −4.3, −4.4. HRMS (ESI) calcd forC₃₄H₅₅BrClN₃O₆Si [M+H]⁺: 742.2576. Found: 742.2648.

S_(N)Ar Precursor (9d)

Following the general reaction protocol (2S,5S,6S)-3d (36.0 g, 68.6mmol, 1.0 equiv) was reacted with 5-bromo-2-chloronicotinoyl chloride 14(35.0 g, 137 mmol, 2.0 equiv) and triethylamine (38.0 mL, 274 mmol, 4.0equiv) in CH₂Cl₂ (500 mL), which provided pure product (2S,5S,6S)-9d(48.5 g, 95%).

(2S,5S,6S)-9d: [α]_(D) ²⁰ −28.4 (c 2.4, CHCl₃). IR (cm⁻¹) 2954, 2930,2856, 1694, 1643, 1513, 1401, 1249, 1155, 1098, 1034. ¹H NMR (500 MHz,CDCl₃, 2:1 mixture of rotamers) δ 8.37 (s, 1H×0.33), 8.32 (s, 1H×0.66),7.70 (d, J=2.5 Hz, 2H×0.33), 7.57 (d, J=2.5 Hz, 2H×0.66), 7.25 (d, J=8.5Hz, 2H, x 0.33), 7.16 (d, J=8.5 Hz, 2H×0.66), 6.87 (d, J=8.5 Hz,2H×0.66), 6.80 (d, J=8.5 Hz, 2H×0.33), 4.55-4.43 (m, 2H×0.33), 4.35-4.25(m, 2H×0.66), 4.02-3.96 (m, 1H), 3.76 (3H, s) 3.71-3.54 (2H, m), 3.48(dd, J=8.5, 5.0 Hz, 1H), 3.25-3.16 (m, 2H), 3.04-2.92 (m, 2H), 2.88-2.85(m, 3H), 2.80-2.73 (m, 1H), 1.44 (s, 9H), 1.13-0.98 (m, 3H), 0.90 (s,9H), 0.08 (s, 6H). ¹³C NMR (125 MHz, CDCl₃, reported as a mixture ofrotamers) δ 167.4, 165.8, 159.5, 159.3, 150.5, 150.2, 145.5, 145.0,142.0, 134.8, 130.3, 129.7, 129.5, 129.3, 119.3, 119.2, 114.1, 113.9,113.8, 79.7, 79.3, 73.1, 72.8, 72.2, 71.0, 70.8, 55.3, 55.3, 52.5, 51.9,44.9, 44.8, 36.0, 34.9, 34.7, 28.6, 28.5, 26.0, 25.6, 18.1, 17.7, 15.7,14.3, 12.2, −4.3. HRMS (ESI) calcd for C₃₄H₅₅BrClN₃O₆Si [M+H]⁺:742.2576. Found: 742.2646.

General Protocol for the S_(N)Ar Cycloetherification (S_(N)Ar-Pyr)

Protocol A:

Cesium fluoride (5 equiv) was added to a solution of amide 9 (1.0 equiv)in DMF (0.1 M) at RT under N₂. The reaction was heated to 85° C. andstirred until complete consumption of starting material was observed(8-12 h). The reaction was cooled and quenched with a saturated NH₄Clsolution. The resulting mixture was extracted with EtOAc. The combinedorganic extracts were dried over MgSO₄, filtered and concentrated. Theresulting crude product was dissolved in THF before NaH (1.0 equiv) wasadded. The mixture was stirred at 60° C. until the reaction was completeby LC/MS (˜1 h). The mixture was cooled and quenched with a saturatedNH₄Cl solution. The resulting mixture was extracted with EtOAc. Thecombined organic extracts were dried over MgSO₄, filtered andconcentrated to provide desired 10 in >95% purity. The material was usedwithout further purification.

Protocol B:

Tetrabutylammonium fluoride (5 equiv) was added to a solution of amide 9(1.0 equiv) in DMF (0.1 M) at 65° C. under N₂. The reaction wasmonitored by LC/MS (7-8 h) and upon completion, cooled and concentrated.The resulting crude product was taken up in a saturated NH₄Cl solutionand extracted with EtOAc. The combined organic extracts were dried overMgSO₄, filtered and concentrated. Flash chromatography on silica gel(gradient: 0% to 30% EtOAc in hexanes) provided cyclic product 10.

Eight-Atom Membered Lactam (10a)

Following protocol A, (2R,5S,6R)-9a (20.9 g, 28.1 mmol, 1.0 equiv) wasreacted with cesium fluoride (21.4 g, 141 mmol, 5.0 equiv) in DMF (280mL), followed by reaction with NaH (60% dispersion in oil, 1.2 g, 28.1mmol, 1.0 equiv) in THF (280 mL) which provided product (2R,5S,6R)-10a(16.4 g, 98%).

(2R,5S,6R)-10a: [α]_(D) ²⁰ +37.7 (c 1.0, CHCl₃). IR (cm⁻¹) 2974, 1690,1627, 1424, 1246, 1153. ¹H NMR (500 MHz, DMSO-d₆, 110° C.) δ 8.23 (d,J=2.5 Hz, 1H), 7.97 (d, J=2.5 Hz, 1H), 7.25 (d, J=8.5 Hz, 2H), 6.90 (d,J=8.5 Hz, 2H), 4.68 (t, J=5.5 Hz, 1H), 4.49 (m, 1H), 4.45 (s, 3H), 3.76(s, 3H), 3.73 (dd, J=10, 7 Hz, 1H), 3.51 (dd, J=10, 5.5 Hz, 1H), 3.44(dd, J=15.5, 7 Hz, 1H), 3.39 (dd, J=15.5, 5.5 Hz, 1H), 3.01 (dd, J=12.5,12 Hz, 1H), 2.79 (s, 3H), 2.06 (m, 1H), 1.36 (s, 9H), 1.24 (d, J=6.5 Hz,3H), 0.93 (d, J=6.5 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆, 110° C.) δ165.6, 158.5, 158.4, 154.3, 149.8, 144.0, 129.8, 128.5, 128.4, 113.4,110.6, 78.5, 74.6, 71.4, 70.5, 54.7, 51.8, 50.0, 48.4, 34.6, 32.2, 27.5,14.0, 9.7. HRMS (ESI) calcd for C₂₈H₃₉BrN₃O₆ [M+H]⁺: 592.2017. Found:592.2040.

Eight-Atom Membered Lactam (10b)

Following protocol A, (2S,5S,6R)-9b (29.2 g, 39.3 mmol, 1.0 equiv) wasreacted with cesium fluoride (29.8 g, 196 mmol, 5.0 equiv) in DMF (400mL), followed by reaction with NaH (60% dispersion in oil, 1.6 g, 39.3mmol, 1.0 equiv) in THF (400 mL) which provided product (2S,5S,6R)-10b(22.4 g, 96%).

(2S,5S,6R)-10b: [α]_(D) ²⁰ +33.7 (c 1.0, CHCl₃). IR (cm⁻¹) 2930, 1682,1628, 1426, 1247, 1153. ¹H NMR (500 MHz, DMSO-d₆, 110° C.) δ 8.32 (d,J=2.5 Hz, 1H), 7.88 (d, J=2.5 Hz, 1H), 7.21 (d, J=8.5 Hz, 2H), 6.88 (d,J=8.5 Hz, 2H), 4.69 (m, 1H), 4.44 (d, J=12 Hz, 1H), 4.41 (d, J=12 Hz,1H), 3.76 (s, 3H), 3.66 (dd, J=10.5, 8 Hz, 1H), 3.51 (m, 2H), 3.40 (m,2H), 2.96 (m, 2H), 2.85 (s, 3H), 2.03 (m, 1H), 1.36 (s, 9H), 1.22 (d,J=6.5 Hz, 1H), 0.94 (d, J=6.5 Hz, 1H). ¹³C NMR (125 MHz, DMSO-d₆, 110°C.) δ 165.9, 158.5, 158.2, 154.2, 149.7, 143.8, 129.9, 128.3, 128.3,113.3, 110.6, 78.5, 74.5, 71.3, 70.1, 54.7, 50.2, 50.1, 47.3, 34.9,34.0, 27.4, 14.0, 9.7. HRMS (ESI) calcd for C₂₈H₃₉BrN₃O₆ [M+H]⁺:592.2017. Found: 592.1998.

Eight-Atom Membered Lactam (10c)

Following protocol B, (2R,5S,6S)-9c (34.0 g, 45.7 mmol, 1.0 equiv) wasreacted with tetrabutylammonium fluoride (1.0 M solution in THF, 229 mL,229 mmol, 5.0 equiv) in DMF (460 mL) which provided product(2R,5S,6S)-10c (21.4 g, 84%).

(2R,5S,6S)-10c: [α]_(D) ²⁰ +32.3 (c 1.0, CHCl₃). IR (cm⁻¹) 2974, 1676,1632, 1434, 1246, 1151. ¹H NMR (500 MHz, DMSO-d₆, 110° C.) δ 8.46 (d,J=2.5 Hz, 1H), 7.96 (d, J=2.5 Hz, 1H), 7.26 (d, J=8.5 Hz, 2H), 6.91 (d,J=8.5 Hz, 2H), 4.49 (d, J=12 Hz, 1H), 4.45 (d, J=12 Hz, 1H), 4.42 (m,1H), 4.04 (ddd, J=10.5, 8, 2.5 Hz, 1H), 3.77 (s, 3H), 3.76 (dd, J=7.5, 7Hz, 1H), 3.64 (dd, J=14.5, 2 Hz, 1H), 3.60 (dd, J=10.5, 5.5 Hz, 1H),3.25 (m, 1H), 3.19 (m, 1H), 3.06 (dd, J=16, 2 Hz, 1H), 2.98 (s, 3H),2.02 (m, 1H), 1.43 (s, 9H), 1.26 (d, J=6.5 Hz, 3H), 0.81 (d, J=6.5 Hz,1H). ¹³C NMR (125 MHz, DMSO-d₆, 110° C.) δ 164.7, 158.5, 154.5, 150.5,140.2, 129.9, 128.4, 125.6, 114.7, 113.4, 87.1, 78.1, 71.5, 70.9, 54.6,51.6, 50.5, 35.3, 35.2, 34.8, 27.6, 14.7, 13.9. HRMS (ESI) calcd forC₂₈H₃₉BrN₃O₆ [M+H]⁺: 592.2017. Found: 592.1989.

Eight-Atom Membered Lactam (10d)

Following protocol B (2S,5S,6S)-9d (36.0 g, 48.4 mmol, 1.0 equiv) wasreacted with tetrabutylammonium fluoride (1.0 M solution in THF, 242 mL,242 mmol, 5.0 equiv) in DMF (480 mL) which provided product(2S,5S,6S)-10d (21.5 g, 80%).

(2S,5S,6S)-10d: [α]_(D) ²⁰ +43.9 (c 1.0, CHCl₃). IR (cm⁻¹) 2974, 1688,1632, 1434, 1246, 1151. ¹H NMR (500 MHz, DMSO-d₆, 110° C.) δ 8.47 (d,J=2.5 Hz, 1H), 7.91 (d, J=2.5 Hz, 1H), 7.25 (d, J=8.5 Hz, 2H), 6.90 (d,J=9 Hz, 2H), 4.46 (q, J=11.5, 20 Hz, 2H), 4.43 (m, 1H), 4.04 (m, 1H),3.73 (dd, J=7, 10 Hz, 1H), 3.66 (dd, J=2.5, 15 Hz, 1H), 3.60 (dd, J=5.5,10 Hz, 1H), 3.29 (dd, J=8, 15 Hz, 1H), 3.20 (dd, J=10.0, 16.5 Hz, 1H),3.10 (dd, J=1.5, 16.5 Hz, 1H), 2.03 (m, 1H), 1.33 (d, J=7 Hz, 3H), 0.83(d, J=7.0 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆, 110° C.) δ 164.7, 159.9,158.5, 154.5, 150.4, 140.2, 130.0, 128.4, 128.3, 125.7, 114.7, 113.3,87.0, 78.5, 71.4, 70.1, 54.7, 51.6, 51.4, 50.5, 35.6, 27.7, 14.8, 14.7.HRMS (ESI) calcd for C₂₈H₃₉BrN₃O₆ [M+H]⁺: 592.2017. Found: 592.1989.

Elaboration of S_(N)Ar Compounds to Final Core (S_(N)Ar-Pyr)

2,6-Lutidine (4.0 equiv) and TBSOTf (3.0 equiv) were added to a solutionof lactam 10 (1.0 equiv) in CH₂Cl₂ (0.1 M) at RT. The mixture wasstirred until complete consumption of starting material was observed byLCMS (<2 h). The reaction was quenched with saturated NH₄Cl solution andextracted with EtOAc. The combined organic extracts were dried overMgSO₄, filtered and concentrated to give the crude silyl carbamate. Theresulting oil was dissolved in THF (0.2 M) before HF.pyridine (70%, 1.0equiv) was added. The mixture was stirred for 40 min, quenched withsaturated NaHCO₃ solution and extracted with EtOAc. The combined organicextracts were dried over MgSO₄, filtered and concentrated to provide thesecondary amine, which was used without any further purification.

The secondary amine (1.0 equiv) was taken up in 1,4-dioxane (0.15 M) andan excess of a 10% NaHCO₃ solution was added. The reaction was cooled to0° C. before a solution of FmocCl (1.2 equiv) in minimal 1,4-dioxane wasadded. The reaction was stirred at RT until no more starting materialwas observed (˜1 h) and reaction was quenched with a saturated NH₄Clsolution. The resulting mixture was extracted with EtOAc. The combinedorganic extracts were dried over MgSO₄, filtered and concentrated. Flashchromatography on silica gel (gradient: 30% to 50% EtOAc in hexanes)provided the desired product 11.

Fmoc Protected Lactam (11a)

Following the general reaction protocol, (2R,5S,6R)-10a (16.7 g, 28.1mmol, 1.0 equiv) was reacted with TBSOTf (19.4 mL, 84.0 mmol, 3.0 equiv)and 2,6-Lutidne (13.1 mL, 112 mmol, 4.0 equiv) in CH₂Cl₂ (300 mL)followed by HF.pyr (3.5 mL, 28.1 mmol, 1.0 equiv) in THF (150 mL). Fmocprotection was carried out using FmocCl (8.7 g, 33.7 mmol, 1.2 equiv)and a 10% NaHCO₃ solution (50 mL) in 1,4-dioxane (200 mL) which providedpure product (2R,5S,6R)-11a (17.5 g, 87% over 2 steps).

(2R,5S,6R)-11a: [α]_(D) ²⁰ +33.5 (c 1.0, CHCl₃). IR (cm⁻¹) 2936, 1698,1627, 1424, 1246, 1165. ¹H NMR (500 MHz, DMSO-d₆, 110° C.) δ 8.31 (d,J=2.5 Hz, 1H), 7.97 (d, J=2.5 Hz, 1H), 7.82 (dd, J=12.5, 6.5 Hz, 2H),7.56 (dd, J=12.5, 6.5 Hz, 2H), 7.38 (td, J=7.5 Hz, 2H), 7.27 (td, J=7.5Hz, 2H), 7.21 (d, J=8.5 Hz, 2H), 6.88 (d, J=8.5 Hz, 2H), 4.68 (dd,J=10.5, 5 Hz, 1 Hz), 4.50 (m, 1H), 4.44 (d, J=12 Hz, 2H). 4.42 (d, J=12Hz, 2H), 4.30 (dd, J=10.5, 6.5 Hz, 1H), 4.24 (dd, J=12.5, 6.5 Hz, 1H),3.75 (s, 3H), 3.70 (dd, J=10.5, 7 Hz, 1H), 3.49 (m, 1H), 3.36 (dd,J=15.5, 5.5 Hz, 1H), 3.00 (dd, J=15.5, 15 Hz, 1H), 2.94 (s, 3H), 2.00(m, 1H), 1.20 (d, J=6.5 Hz, 3H), 0.90 (d, J=6.5 Hz, 3H). ¹³C NMR (125MHz, DMSO-d₆, 110° C., reported as a mixture of rotamers) δ 165.9,165.6, 158.5, 158.3, 155.0, 149.8, 149.6, 143.9, 143.8, 143.3, 142.3,140.3, 140.2, 139.0, 137.0, 129.9, 128.4, 128.2, 126.8, 126.6, 126.3,124.2, 120.6, 119.3, 119.2, 118.6, 113.4, 110.2, 108.2, 75.7, 71.4,70.5, 66.2, 54.7, 52.5, 50.1, 50.0, 47.8, 47.7, 46.5, 35.3, 34.3, 33.4,33.2, 13.9, 9.8, 9.6. HRMS (ESI) calcd for C₃₈H₄₁BrN₃O₆ [M+H]⁺:714.2179. Found: 714.2187.

Fmoc Protected Lactam (11b)

Following the general reaction protocol (2S,5S,6R)-10b (23.3 g, 39.3mmol, 1.0 equiv) was reacted with TBSOTf (27.1 mL, 118 mmol, 3.0 equiv)and 2,6-Lutidne (18.3 mL, 157 mmol, 4.0 equiv) in CH₂Cl₂ (400 mL)followed by HF.pyr (4.9 mL, 39.3 mmol, 1.0 equiv) in THF (200 mL). Fmocprotection was carried out using FmocCl (12.2 g, 47.2 mmol, 1.2 equiv)and a 10% NaHCO₃ solution (50 mL) in 1,4-dioxane (250 mL) which providedpure product (2S,5S,6R)-11b (24.7 g, 88% over 2 steps).

(2S,5S,6R)-11b: [α]²⁰ +28.0 (c 1.0, CHCl₃). IR (cm⁻¹): 2935, 1701, 1630,1427, 1292, 1248, 759, 719. ¹H NMR (500 MHz, DMSO-d₆, 110° C.) δ 8.32(d, J=2.5 Hz, 1H), 7.90 (d, J=2.5 Hz, 1H), 7.83 (dd, J=7.5, 5.0 Hz, 2H),7.57 (t, J=8.4 Hz, 2H), 7.41-7.37 (m, 2H), 7.31-7.24 (m, 2H), 7.21 (d,J=8.5 Hz, 2H), 6.88 (d, J=8.5 Hz, 2H), 4.70-4.64 (m, 1H), 4.60-4.51 (m,1H), 4.46-4.40 (m, 1H), 4.42 (d, J=4.4 Hz, 2H), 4.34-4.31 (m, 1H), 4.25(t, J=6.4 Hz, 1H), 3.76 (s, 3H), 3.68 (dd, J=10.0, 8.0 Hz, 1H), 3.53 (q,J=5.0 Hz, 2H), 3.51-3.47 (m, 1H), 3.46-3.35 (m, 2H), 2.97 (dd, J=15.0,7.5 Hz, 1H), 2.83 (s, 3H), 2.03-1.96 (m, 1H), 1.19 (d, J=6.9 Hz, 3H),0.90 (d, J=6.6, 3H). ¹³C NMR (125 MHz, DMSO-d₆, 110° C., reported as amixture of rotamers) δ 167.1, 159.8, 159.5, 156.3, 151.04, 151.00,145.1, 144.66, 144.63, 141.58, 141.55, 131.2, 129.6, 129.5, 128.2,127.6, 125.4, 120.6, 114.6, 112.0, 95.0, 75.7, 72.6, 71.3, 67.5, 56.0,55.9, 52.3, 51.5, 49.1, 47.8, 35.84, 35.78, 35.2, 15.4, 10.9. HRMS (ESI)calcd for C₃₈H₄₁BrN₃O₆[M+H]⁺: 714.2179. Found: 714.2178.

Fmoc Protected Lactam (11c)

Following the general reaction protocol (2R,5S,6S)-10c (19.0 g, 32.1mmol, 1.0 equiv) was reacted with TBSOTf (22.1 mL, 96.0 mmol, 3.0 equiv)and 2,6-Lutidne (14.9 mL, 128 mmol, 4.0 equiv) in CH₂Cl₂ (320 mL)followed by HF.pyr (4.0 mL, 32.1 mmol, 1.0 equiv) in THF (160 mL). Fmocprotection was carried out using FmocCl (10.0 g, 38.5 mmol, 1.2 equiv)and a 10% NaHCO₃ solution (50 mL) in 1,4-dioxane (200 mL) which providedpure product (2R,5S,6S)-11c (20.9 g, 91% over 2 steps).

(2R,5S,6S)-11c: [α]_(D) ²⁰ +27.9 (c 1.0, CHCl₃). IR (cm⁻¹) 2936, 1699,1633, 1435, 1427, 1171, 1086, 759, 741. ¹H NMR (500 MHz, DMSO-d₆, 110°C.) α 8.44 (s, 1H), 7.96 (s, 1H), 7.83 (d, J=6.5 Hz, 2H), 7.64 (t, J=6.5Hz, 2H), 7.39 (dd, J=16.5, 7.5 Hz, 2H), 7.30 (t, J=7.5 Hz, 2H), 7.25 (d,J=8.1 Hz, 2H), 6.89 (d, J=8.1 Hz, 2H), 4.48-4.41 (m, 5H), 4.30-4.25 (m,1H), 3.98-3.88 (m, 1H), 3.76 (s, 3H), 3.74-3.70 (m, 1H), 3.62-3.51 (m,2H), 3.34-3.24 (m, 1H), 3.18-3.10 (m, 1H), 3.05 (s, 1H), 3.02 (s, 3H),2.04-1.93 (m, 1H), 1.24 (d, J=6.8 Hz, 3H), 0.68 (s, 3H). ¹³C NMR (125MHz, DMSO-d₆, 110° C., reported as a mixture of rotamers) δ 164.71,164.67, 160.1, 159.8, 158.5, 155.1, 150.5, 150.4, 143.4, 142.3, 140.4,140.3, 139.0, 137.0, 129.92, 129.88, 128.4, 128.2, 126.83, 126.80,126.5, 126.3, 125.6, 124.2, 120.5, 119.2, 119.1, 114.8, 114.6, 113.3,108.1, 87.4, 86.7, 71.5, 70.9, 70.8, 65.9, 54.6, 54.1, 51.7, 51.6, 51.4,50.7, 50.5, 46.6, 35.3, 35.0, 34.9, 34.7, 15.1, 14.7, 13.86, 13.85. HRMS(ESI) calcd for C₃₈H₄₁BrN₃O₆ [M+H]⁺: 714.2179. Found: 714.2180.

Fmoc Protected Lactam (11d)

Following the general reaction protocol (2S,5S,6S)-10d (21.0 g, 35.4mmol, 1.0 equiv) was reacted with TBSOTf (24.4 mL, 106 mmol, 3.0 equiv)and 2,6-Lutidne (16.5 mL, 142 mmol, 4.0 equiv) in CH₂Cl₂ (350 mL)followed by HF.pyr (4.4 mL, 35.4 mmol, 1.0 equiv) in THF (175 mL). Fmocprotection was carried out using FmocCl (11.0 g, 42.4 mmol, 1.2 equiv)and a 10% NaHCO₃ solution (50 mL) in 1,4-dioxane (250 mL) which providedpure product (2S,5S,6S)-11d (25.2 g, 99% over 2 steps).

(2S,5S,6S)-11d: [α]²⁰+47.0 (c 1.0, CHCl₃). IR (cm⁻¹): 2936, 1699, 1634,1436, 1248, 758, 742. ¹H NMR (500 MHz, DMSO-d₆, 110° C., 8:1 mixture ofrotamers, only the major rotamer reported) δ 8.45 (s, 1H), 7.94-7.91 (m,1H), 7.85-7.83 (m, 2H), 7.65 (t, J=7.5 Hz, 2H), 7.41-7.39 (m, 2H),7.33-7.27 (m, 2H), 7.24 (d, J=8.5 Hz, 2H), 6.90 (d, J=8.6 Hz, 2H),4.51-4.37 (m, 5H), 4.27 (t, J=5.5 Hz, 1H), 3.98-3.88 (m, 1H), 3.77 (s,3H), 3.75-3.68 (m, 1H), 3.60 (dd, J=10.5, 5.5 Hz, 1H), 3.59-3.52 (m,1H), 3.37-3.26 (m, 1H), 3.17-3.05 (m, 2H), 3.02 (s, 3H), 2.03-1.91 (m,1H), 1.31 (d, J=7.0 Hz, 3H), 0.77-0.63 (m, 3H). ¹³C NMR (125 MHz,DMSO-d₆, 110° C., reported as a mixture of rotamers) δ 166.0, 165.8,161.1, 159.8, 156.5, 151.9, 151.8, 151.7, 151.6, 144.8, 141.6, 141.5,132.0, 131.4, 129.7, 129.6, 128.2, 128.1, 127.7, 127.6, 127.0, 125.5,125.4, 120.6, 116.1, 116.0, 114.7, 114.6, 88.0, 87.9, 72.7, 71.4, 71.3,67.2, 65.1, 56.0, 55.9, 52.8, 52.7, 51.85, 47.9, 37.5, 37.1, 36.3, 29.6,16.1, 16.0, 15.9. HRMS (ESI) calcd for C₃₈H₄₁BrN₃O₆ [M+H]⁺: 714.2179.Found: 714.2177.

Lactam 11 (1.0 equiv) was dissolved in a 5:1 solution of CH₂Cl₂ and pH 7buffer solution (0.15 M). The mixture was cooled to 0° C. and DDQ (1.2equiv) was added. The mixture was stirred for 10 min at 0° C. and anadditional 1 h at RT before being quenched with a saturated NaHCO₃solution and filtered through a ceramic Buchner funnel equipped withfilter paper. The organic and aqueous layers were separated and theaqueous layer extracted with CH₂Cl₂. The combined organic extracts werewashed with saturated NaHCO₃ solution, dried over MgSO₄, filtered andconcentrated. Flash chromatography on silica gel (50% to 100% EtOAc inhexanes) gave pure product 29 as a white foaming solid.

S_(N)Ar-Pyr Final Core (29a)

Following the general reaction protocol (2R,5S,6R)-11a (17.4 g, 24.4mmol, 1.0 equiv) was reacted with DDQ (6.6 g, 29.2 mmol, 1.2 equiv) inCH₂Cl₂ (150 mL) and pH 7 buffer (30 mL) to give product (2R,5S,6R)-29a(14.0 g, 97%).

(2R,5S,6R)-29a: [α]_(D) ²⁰ +29.0 (c 1.0, CHCl₃). IR (cm⁻¹) 3430, 3052,2972, 1696, 1623, 1423, 1290, 1164. ¹H NMR (500 MHz, DMSO-d₆, 110° C.) δ8.31 (d, J=2.5 Hz, 1H), 7.98 (d, J=2.5 Hz, 1H), 7.84 (t, J=7.5 Hz, 2H),7.59 (t, J=7.5 Hz, 2H), 7.38 (m, 2H), 7.28 (m, 2H), 4.76 (t, J=2 Hz,1H), 4.50 (t, J=2 Hz, 1H), 4.37 (dd, J=6.5, 6 Hz, 1H), 4.30 (m, 3H),3.64 (m, 1H), 3.51 (m, 3H), 3.36 (dd, J=15.5, 5.5 Hz, 1H), 3.00 (dd,J=14, 12.5 Hz, 1H), 2.95 (s, 3H), 2.84 (s, 3H), 2.10 (m, 1H), 1.18 (d,J=6.5 Hz, 3H), 0.91 (d, J=6.5 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆, 110°C.) δ 165.7, 158.2, 155.0, 149.7, 144.0, 143.4, 140.2, 126.9, 126.8,126.4, 124.3, 124.2, 119.3, 74.3, 66.2, 62.3, 54.1, 50.2, 48.5, 46.5,34.4, 33.1, 13.5, 9.7. HRMS (ESI) calcd for C₃₀H₃₃BrN₃O₅ [M+H]:594.1598. Found: 594.1570.

(2S,5R,6S)-ent-29a: [α]_(D) ²⁰ −32.4 (c 1.0, CHCl₃).

S_(N)Ar-Pyr Final Core (29b)

Following the general reaction protocol (2S,5S,6R)-11b (24.7 g, 34.6mmol, 1.0 equiv) was reacted with DDQ (9.4 g, 41.5 mmol, 1.2 equiv) inCH₂Cl₂ (200 mL) and pH 7 buffer (40 mL) to give product (2S,5S,6R)-29b(19.0 g, 92%).

(2S,5S,6R)-29b: [α]²⁰ +30.1 (c 1.0, CHCl₃). IR (cm⁻¹) 3435 (br), 2971,1696, 1625, 1426, 1291, 757. ¹H NMR (500 MHz, DMSO-d₆, 110° C.) δ 8.32(d, J=2.5 Hz, 1H), 7.96 (d, J=2.5 Hz, 1H), 7.84 (dd, J=7.5, 4.6 Hz, 2H),7.61-7.56 (m, 2H), 7.38 (dt, J=7.5, 5.0 Hz, 2H), 7.33-7.25 (m, 2H), 4.73(br s, 1H), 4.46 (dd, J=10.6, 6.3 Hz, 1H), 4.41 (br s, 1H), 4.34 (dd,J=10.5, 6.3 Hz, 2H), 4.26 (t, J=6.3 Hz, 1H), 3.71-3.66 (m, 1H), 3.54(dd, J=10.9, 5.0 Hz, 2H), 3.50-3.39 (m, 2H), 3.02 (dd, J=15.4, 12.4 Hz,1H), 2.94 (s, 1H), 2.86 (s, 3H), 2.10-1.99 (m, 1H), 1.19 (d, J=6.9 Hz,3H), 0.93 (d, J=6.6 Hz, 3H). ¹³C NMR (125 MHz, CDCl₃, 60° C., reportedas a mixture of rotamers) δ 167.3, 159.3, 156.8, 151.1, 145.6, 144.2.144.1, 141.6, 141.5, 127.9, 127.8, 127.2, 125.1, 125.0, 120.11, 120.08,119.5, 112.4, 95.0, 76.9, 67.8, 63.3, 60.3, 53.0, 47.6, 36.6, 34.3,14.9, 10.6. HRMS (ESI) calcd for C₃₀H₃₃BrN₃O₅ [M+H]⁺: 594.1598. Found:594.1599.

(2R,5R,6S)-ent-29b: [α]_(D) ²⁰ −32.4 (c 1.0, CHCl₃).

S_(N)Ar-Pyr Final Core (29c)

Following the general reaction protocol (2R,5S,6S)-11c (20.9 g, 29.2mmol, 1.0 equiv) was reacted with DDQ (8.0 g, 35.0 mmol, 1.2 equiv) inCH₂Cl₂ (175 mL) and pH 7 buffer (35 mL) to give product (2R,5S,6S)-29c(16.1 g, 93%).

(2R,5S,6S)-29c: [α]_(D) ²⁰ + 28.3 (c 1.0, CHCl₃). IR (cm⁻¹) 3436 (br),2936, 1694, 1626, 1435, 758. ¹H NMR (500 MHz, DMSO-d₆, 110° C.) δ 8.45(s, 1H), 7.99 (d, J=2.4 Hz, 1H), 7.84 (d, J=7.2 Hz, 2H), 7.66 (t, J=7.4Hz, 2H), 7.39 (dd, J=16.0, 7.2 Hz, 2H), 7.33-7.25 (m, 2H), 4.50-4.40 (m,2H), 4.46 (br s, 1H), 4.29 (t, J=6.0 Hz, 1H), 4.17 (q, J=6.5 Hz, 1H),4.02-3.89 (m, 1H), 3.78-3.72 (m, 1H), 3.65-3.55 (m, 2H), 3.44-3.36 (m,1H), 3.19-3.14 (m, 1H), 3.10-3.05 (m, 1H), 3.05 (s, 3H), 2.08 (s, 1H),1.25 (d, J=6.7 Hz, 3H), 0.73 (s, 3H). ¹³C NMR (125 MHz, CDCl₃, 60° C.,reported as a mixture of rotamers) δ 167.1, 161.2, 152.2, 144.3, 141.69,141.65, 141.59, 127.8, 127.22, 127.18, 125.3, 120.1, 116.4, 88.3, 67.9,65.3, 57.3, 54.1, 53.1, 47.7, 36.9, 35.3, 16.3, 15.8, 14.4, 14.3. HRMS(ESI) calcd for C₃₀H₃₃BrN₃O₅ [M+H]⁺: 594.1598. Found: 594.1604.

(2S,5R,6R)-ent-29c: [α]_(D) ²⁰ −26.6 (c 1.0, CHCl₃).

S_(N)Ar-Pyr Final Core (29d)

Following the general reaction protocol (2S,5S,6S)-11d (25.2 g, 35.3mmol, 1.0 equiv) was reacted with DDQ (9.6 g, 42.3 mmol, 1.2 equiv) inCH₂Cl₂ (200 mL) and pH 7 buffer (40 mL) to give product (2S,5S,6S)-29d(20.0 g, 95%).

(2S,5S,6S)-29d: [α]²⁰ +38.0 (c 1.0, CHCl₃). IR (cm⁻¹): 3435 (br), 2937,1693, 1624, 1435, 757. ¹H NMR (500 MHz, DMSO-d₆, 110° C.) δ 8.45 (s,1H), 7.97 (s, 1H), 7.84 (d, J=7.2 Hz, 2H), 7.66 (t, J=7.2 Hz, 2H),7.43-7.35 (m, 2H), 7.35-7.25 (m, 2H), 4.51-4.42 (m, 2H), 4.38-4.33 (m,1H), 4.31-4.21 (m, 2H), 3.99-3.88 (m, 1H), 3.69-3.55 (m, 3H), 3.44-3.35(m, 1H), 3.14 (s, 2H), 3.04 (s, 3H), 2.00 (s, 1H), 1.29 (d, J=6.9 Hz,3H), 0.73 (s, 3H). ¹³C NMR (125 MHz, CDCl₃, 60° C.) δ 167.3, 161.0,157.0, 152.2, 144.3, 141.6, 141.6, 127.8, 127.2, 127.2, 125.3, 120.1,116.5, 88.4, 67.9, 64.6, 57.3, 53.4, 53.2, 47.7, 36.9, 36.1, 16.1, 14.4.HRMS (ESI) calcd for C₃₀H₃₃BrN₃O₅ [M+H]⁺: 594.1598. Found: 594.1603.

(2R,5R,6R)-ent-29d: [α]_(D) ²⁰ −36.4 (c 1.0, CHCl₃).

Solid-Phase Library Synthesis

General Methods:

Solid-phase synthesis was conducted on silicon-functionalizedpolystyrene SynPhase™ Lanterns (L-series) equipped with radio frequencytransponders (TranStems) for AccuTag directed sorting and compoundtracking. Quality-control Lanterns were included at each synthesis stepfor reaction monitoring by UPLC (UV 210 nM) after HF-cleavage. Allreactions were conducted in heavy wall pressure vessels from ChemGlasswith agitation in New Brunswick Scientific incubator shakers.

Scaffold Loading:

To a flame-dried flask containing silicon-functionalized Lanterns wasadded a freshly prepared solution of TfOH in anhydrous DCM (9.0 equiv, 5g of TfOH/100 mL of DCM) was added. Each flask was shaken at RT for 10min at which time the Lanterns had turned bright orange. The deep redTfOH solution was removed via cannula and anhydrous 2,6-lutidine (12.0equiv relative to Si) was added. Once the Lantern color had changed fromorange to white, the scaffold (1.2 equiv. relative to Si) was added as asolution in anhydrous DCM (0.4 mL/Lantern) and the reaction mixture wasshaken for 48 h overnight. The loading mixture was removed and set aside(to recover any unreacted alcohol) and the Lanterns were washed with thefollowing solvents for 30 min intervals: DCM, THF, 3:1 THF/IPA, 3:1THF/H₂O, DMF, 3:1 THF/H₂O, 3:1 THF/IPA, THF, DCM. The Lanterns were thendried on a lyophilizer overnight prior to sorting. All 8 stereoisomersof 29 were loaded via the same protocol.

Fmoc Removal:

To a flask containing Lanterns was added a solution of 20% piperidine inDMF (0.8 mL/Lantern). After shaking at RT for 30 min, the piperidinesolution was removed and the Lanterns were washed with the followingsolvents for 30 min intervals: DMF, 3:1 THF/H₂O, 3:1 THF/IPA, THF, DCM.The Lanterns were then dried on a lyophilizer overnight prior tosorting.

N-Capping/Isocyanates:

To each flask containing Lanterns was added DCM (0.8 mL/Lantern)followed the desired isocyanate (15 equiv). The Lanterns were shaken atRT overnight and then washed with following solvents for 30 minintervals: DCM, DMF, 3:1 THF/H₂O, 3:1 THF/IPA, THF, DCM. The Lanternswere then dried on a lyophilizer overnight prior to sorting.

Cross-Coupling/Suzuki:

To each flask containing lanterns was added ethanol (0.800 mL/lantern)followed by the desired boronic acid (20 equiv), triethylamine (40equiv) and Pd(PPh₃)₂Cl₂ (1 equiv). The resulting mixture was degassedwith a stream of N₂ before shaking at 60° C. After 4 days, the reactionmixture was removed and the Lanterns were washed with following solventsfor 30 min intervals: DCM, DMF, NaCN solution (0.1M) in 1:1 THF/H₂O,DMF, 3:1 THF/H₂O, 3:1 THF/IPA, THF, DCM. The lanterns were then dried ona lyophilizer overnight prior to sorting.

Cross-Coupling/Sonogashira:

To each flask containing lanterns was added DMF (0.800 mL/lantern)followed by the desired alkyne (20 equiv), CuI (3 equiv),diisopropylethylamine (30 equiv) and Pd(PPh₃)₂Cl₂ (1 equiv). Theresulting mixture was degassed with a stream of N₂ before shaking at 60°C. overnight. After 24 h, the reaction mixture was removed and theLanterns were washed with following solvents for 30 min intervals: DCM,DMF, NaCN solution (0.1 M) in 1:1 THF/H₂O (30 min), DMF, 3:1 THF/H₂O,3:1 THF/IPA, THF, DCM. The lanterns were then dried on a lyophilizerovernight prior to sorting.

Cleavage Protocol:

To a 96-well plate containing Lanterns was added a 15% solution ofHF/pyridine in stabilized THF (350 μL/Lantern). After 2 h the cleavagesolution was quenched with TMSOMe (700 μL/Lantern) and the contents ofeach well were transferred to a pre-weighed 2-mL vial. The Lanterns werewashed with an additional 200 μL of stabilized THF (or THF/MeOH) and thesolution was transferred to the 2-mL vial. The samples were concentratedon a Genevac® solvent evaporation system overnight without heating.Loading masses for each alcohol was determined on a FlexiWeigh® system.

Compound 106 was synthesized following the general methods for librarysynthesis. Compound 29 was loaded onto Lanterns followed by subsequentFmoc removal. The secondary amine was capped with 4-fluorophenylisocyanate. Suzuki cross coupling was carried out using2-benzofuranylboronic acid. Compound 106 was then cleaved from theLanterns and isolated following the standard cleavage protocol.

Compound 107 was synthesized following the general methods for librarysynthesis. Compound 29 was loaded onto Lanterns followed by subsequentFmoc removal. The secondary amine was capped with 4-fluorophenylisocyanate. Sonogashira cross coupling was carried out using4-fluorophenyl acetylene. Compound 107 was then cleaved from theLanterns and isolated following the standard cleavage protocol.

Preparation of Compound 8 in Table 1:

General Procedure for the Preparation of Azido Alkynes

Step 1:

A solution of linear amine template 3 (1.0 equiv), carboxylic acid 18(1.3 equiv) (Khoukhi, et al., 2000, Tetrahedron 43:1811-1822), andbenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP) (1.1-1.3 equiv) in dichloromethane (0.1-0.2 molar) was cooled to0° C. in a water/ice bath. Then diisopropyl ethylamine (DIEA) (3.0equiv) was added, and the reaction was stirred for 30 minutes beforeremoving the ice bath, and allowing the reaction to stir overnight atRT. The reaction mixture was quenched with water, the layers wereseparated and the CH₂Cl₂ layer was washed twice with water, dried oversodium sulfate, and then filtered through a short plug of silica gel.The crude product was pure enough to be taken on to next step withoutfurther purification.

Step 2:

The crude product from step 1 (1.0 equiv) in THF (0.1 molar) was cooledto 0° C. using an ice bath. Tetrabutylammonium fluoride (TBAF) (1.0 Msolution in THF, 2.0 equiv) was added drop wise and the reaction wasstirred for 14 hours, slowly warming to RT. Analysis of the reaction byLC-MS indicated complete disappearance of the starting material. Thereaction was quenched with saturated aqueous NH₄Cl and THF was removedusing rotavap, the reaction was diluted with EtOAc and the organic layerwas washed with water (acidified to pH=4, using AcOH). This step wasrepeated twice to remove excess TBAF. The combined organic layers weredried over sodium sulfate and concentrated to provide the crude product.Purification of crude product using silica gel provided the purealcohol.

Step 3:

The product from previous step (1.0 equiv) was dissolved in THF/DMF(6:1) (0.2 molar). To this slightly yellow solution was added propargylbromide (10 equiv) and the solution was cooled to −78° C. followed bydropwise addition of 1M NaHMDS (3 equiv). The ice bath was removed andthe above mixture was warmed to RT gradually. The reaction was quenchedwith saturated ammonium chloride and the organic layer was evaporated.The aqueous layer was extracted with EtOAc, and the combined organiclayers were washed with brine, dried over sodium sulfate andconcentrated to yield the crude mixture. The crude mixture was purifiedusing silica gel to yield 19.

Azido-Alkyne (19a)

Following general protocol for step 1, (2R,5S,6R)-(−)-3a (40.7 g, 78mmol 1.0 equiv) was reacted with carboxylic acid 18 (11.09 g, 85 mmol1.1 equiv), PyBOP (44.4 g, 85 mmol, 1.1 equiv) and DIEA (40.6 mL, 233mmol, 3.0 equiv) in dichloromethane (400 mL, 0.18 molar) for 16 h.Workup as in general procedure yielded the crude product, which was usedin the next step without further purification.

Following the general protocol for step 2, the crude product (49.6 g, 78mmol 1.0 equiv) was dissolved in THF (500 mL) and reacted with TBAF (1.0M in THF, 156 mL, 156 mmol, 2 equiv) for 14 h. Workup and purificationprovided the pure alcohol (37.8 g, 93% over 2 steps).

Following the general protocol for step 3, the purified alcohol (25.4 g,48.7 mmol, 1.0 equiv) was dissolved in THF (209 ml) and DMF (34.8 ml)and cooled to −78° C., the solution was treated with propargyl bromide(36.7 mL, 487 mmol, 10 equiv) and NaHMDS (1.0 M in THF, 146 mL, 146mmol, 30 equiv) and warmed to RT over 3 h. Flash chromatography usingsilica (10% to 50% EtOAc in hexanes) provided the pure product 19a(26.11 g, 96% yield).

(2R,5S,6R)-19a: [α]_(D) ²⁰ −7.7 (c 2.8, CHCl₃). IR (cm⁻¹) 3297, 2973,1683, 1421, 1213, 1152. ¹H NMR (300 MHz, CDCl₃, 1.1:1 mixture ofrotamers, asterisk denotes minor rotamer peaks) δ 7.10 (d, J=8.0 Hz,2H)*, 7.07 (d, J=7.7 Hz, 2H), 6.74 (d, J=8.0 Hz, 2H)*, 6.73 (d, J=7.7Hz, 2H), 4.32-4.26 (m, 2H), 4.15-3.95 (m, 2H), 3.65 (s, 3H), 3.57-3.40(m, 1H), 3.28-3.10 (m, 2H), 2.78 (s, 3H)*, 2.70 (s, 3H), 2.50-2.20 (m,3H), 2.10-2.00 (m, 1H), 1.76 (p, J=6.6 Hz, 2H), 1.33 (s, 9H), 1.14 (d,J=6.6 Hz, 3H)*, 1.10 (d, J=6.6 Hz, 3H), 0.85 (d, J=6.6 Hz, 3H)*, 0.73(d, J=6.6 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 173.3, 172.3, 159.2, 159.1,155.7, 155.4, 130.4, 129.8, 129.2, 113.7, 113.6, 80.5, 79.8, 79.7, 79.4,79.2, 74.7, 74.1, 72.7, 72.6, 71.8, 71.3, 57.7, 57.4, 55.1, 54.3, 52.4,50.9, 50.8, 50.5, 50.1, 49.7, 49.4, 43.4, 35.8, 35.2, 34.8, 34.0, 30.9,30.5, 28.4, 24.6, 24.5, 16.3, 15.5, 12.4, 11.5 HRMS (ESI) calcd forC₂₉H₄₅N₅O₆ [M+H]⁺: 560.3443. Found: 560.3445.

Azido-Alkyne (19b)

Following the general protocol for step 1, (2S,5S,6R)-(−)-3b (45.0 g, 86mmol, 1.0 equiv) was reacted with carboxylic acid 18 (12.18 g, 94 mmol,1.1 equiv), PyBOP (49.1 g, 94 mmol, 1.1 equiv) and DIEA (34.8 mL, 258mmol, 3.0 equiv) in dichloromethane (400 mL, 0.2 molar) for 16 h. Workupas in general procedure yielded the crude product, which was used in thenext step without further purification.

Following the general protocol for step 2, crude product (54.5 g, 1.0equiv) was dissolved in THF (700 mL) and reacted with TBAF (1.0 M inTHF, 171 mL, 171 mmol, 2 equiv) for 14 h. Workup and purificationprovided pure alcohol (41.89 g, 94% over 2 steps).

Following the general protocol for step 3, the purified alcohol (25.5 g,48.7 mmol, 1.0 equiv) was dissolved in THF (210 ml) and DMF (35.0 ml)and cooled to −78° C., the solution was treated with propargyl bromide(36.8 mL, 489 mmol, 3.0 equiv) and NaHMDS (1.0 M in THF, 196 mL, 196mmol, 4 equiv) and warmed to RT over 3 h. Flash chromatography usingsilica (10% to 50% EtOAc in hexanes) provided the pure product 19b(25.25 g, 92% yield). (2S,5S,6R)-19b: [α]_(D) ²⁰ −17.4 (c 2.1, CHCl₃).

Azido-Alkyne (19c)

Following general protocol for step 1, (2R,5S,6S)-(+)-3c (26.2 g, 49.9mmol, 1.0 equiv) was reacted with carboxylic acid 18 (7.73 g, 59.9 mmol,1.2 equiv), PyBOP (33.8 g, 64.9 mmol, 1.3 equiv) and DIEA (26.5 mL, 152mmol, 3.0 equiv) in dichloromethane (500 mL, 0.1 molar) for 16 h. Workupas in general procedure yielded the crude product, which was used in thenext step without further purification.

Following the general protocol for step 2, the crude product (31.7 g,49.9 mmol, 1.0 equiv) was dissolved in THF (400 mL) and reacted withTBAF (1.0 M in THF, 100 mL, 100 mmol, 2 equiv) for 14 h. Workup andpurification provided the pure alcohol (20.5 g, 79% over 2 steps).

Following the general protocol for step 3, the purified alcohol (19 g,36.4 mmol, 1.0 equiv) was dissolved in THF (156 ml) and DMF (26 ml) andcooled to −78° C., the solution was treated with propargyl bromide (36.7mL, 487 mmol, 10 equiv) and NaHMDS (1.0 M in THF, 109 mL, 109 mmol, 3.0equiv) and warmed to RT over 3 h. Flash chromatography using silica (10%to 50% EtOAc in hexanes) provided pure product 19c (18.15 g, 91% yield).

(2R,5S,6S)-19c: [α]_(D) ²⁰ +9.9 (c 10.4, CHCl₃).

Azido-Alkyne (19d)

Following general protocol for step 1, (2S,5S,6S)-(+)-3d (35 g, 66.7mmol, 1.0 equiv) was reacted with carboxylic acid 18 (9.47 g, 73.4 mmol,1.1 equiv), PyBOP (38.2 g, 73.4 mmol, 1.1 equiv) and DIEA (34.9 mL, 200mmol, 3.0 equiv) in dichloromethane (400 mL, 0.18 molar) for 16 h.Workup as in general procedure yielded the crude product, which was usedin the next step without further purification.

Following the general protocol for step 2, the crude product (42.4 g,66.7 mmol, 1.0 equiv) was dissolved in THF (400 mL) and reacted withTBAF (1.0 M in THF, 133 mL, 133 mmol, 2 equiv) for 14 h. Workup andpurification provided the pure alcohol (37.8 g, 72% over 2 steps).

Following the general protocol for step 3, the purified alcohol (25.4 g,48.7 mmol, 1.0 equiv) was dissolved in THF (209 ml) and DMF (34.8 ml)and cooled to −78° C., the solution was treated with propargyl bromide(36.7 mL, 487 mmol, 10 equiv) and NaHMDS (1.0 M in THF, 146 mL, 146mmol, 3.0 equiv) and warmed to RT over 3 h. Flash chromatography usingsilica (10% to 50% EtOAc in hexanes) provided the pure product 19d(26.11 g, 96% yield).

(2S,5S,6S)-19d: [α]_(D) ²⁰ −9.8 (c 4.4, CHCl₃).

General Procedure for Cu Catalyzed [3+2] Cycloaddition

The Cu-catalyzed reactions were run in two batches and the crudeproducts were combined and purified to yield the products. To a degassedsolution of the azido alkyne 19 (1.0 equiv) in toluene (0.01 molar) at55° C. was added Amberlyst-21 CuPF₆ (1.0 equiv) (Kelly, et al., 2009,Org. Lett. 11:2257-2260). The reaction was heated at 55° C. andmonitored by LC. After 16 h the reaction was complete, beads werefiltered and the solvent was removed under reduced pressure. Therecovered beads were utilized to run a second batch of the reaction, thecrude product from both batches were combined and purified to yield theproduct.

1,4-Macrocyclic Triazole (7a)

Following the general reaction protocol, 7.1 g and 6.7 g batches of(2R,5S,6R)-19a (1.0 equiv) was reacted with Amberlyst-21 CuPF₆ (70.0 g,0.20 mmol/g, 1.0 equiv) in toluene (1200 mL) for 16 h. Flashchromatography using silica (80% to 100% EtOAc in hexanes) provided thepure product (2R,5S,6R)-7a (8.88 g, 57%). (2R,5S,6R)-7a: [α]_(D) ²⁰ +4.8(c 0.5, CHCl₃). IR (cm⁻¹) 3014, 1674, 1449, 1141, 1058. ¹H NMR (500 MHz,DMSO-d₆, 130° C.) δ 7.92 (s, 1H), 7.25 (d, J=7.6, 2H), 6.93 (d, J=8.4,2H), 4.73 (s, 1H), 4.46-4.43 (m, 2H) 4.42 (s, 2H), 3.78 (s, 3H),3.77-3.68 (m, 2H), 3.59-3.52 (m, 1H), 3.48-3.36 (m, 4H), 3.29-3.23 (m,1H), 3.03-2.97 (m, 2H), 2.97 (s, 3H) 2.52 (s, 1H), 2.30-2.04 (m, 4H),1.85-1.75 (m, 3H), 1.47 (s, 9H) 1.17 (d, J=6.4 Hz, 3H), 0.79 (d, J=6.7Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆, 130° C.) δ 173.2, 160.1, 156.2,145.2, 131.6, 129.8, 126.7, 115.0, 78.8, 73.3, 73.1, 62.6, 56.2, 52.9,50.6, 50.0, 48.2, 36.3, 34.6, 31.0, 29.1, 26.2, 16.3, 12.3. HRMS (ESI)calcd for C₂₉H₄₅N₅O₆ [M+H]⁺: 560.3443. Found: 560.3445.

1,4-Macrocyclic Triazole (7b)

Following the general reaction protocol, 6.4 g and 6.3 g batches of(2S,5S,6R)-19b (1.0 equiv) was reacted with Amberlyst-21 CuPF₆ (70.0 g,0.18 mmol/g, 1.0 equiv) in toluene (1200 ml) for 16 h. Flashchromatography using silica (80% to 100% EtOAc in hexanes) provided thepure product (2S,5S,6R)-7b (7.33 g, 58%).

(2S,5S,6R)-7b: [α]_(D) ²⁰ +6.4 (c 5.7, CHCl₃).

1,4-Macrocyclic Triazole (7c)

Following the general reaction protocol, 5.0 g and 5.1 g batches of(2R,5S,6S)-19c (1.0 equiv) was reacted with Amberlyst-21 CuPF₆ (45.0 g,0.22 mmol/g, 1.0 equiv) in toluene (1200 ml) for 16 h. Flashchromatography using silica (80% to 100% EtOAc in hexanes) provided thepure product (2R,5S,6S)-7c (7.70 g, 76%).

(2R,5S,6S)-7c: [α]_(D) ²⁰ +2.8 (c 4.8, CHCl₃).

1,4-Macrocyclic Triazole (7d)

Following the general reaction protocol, 5.0 g and 5.3 g batches of(2S,5S,6S)-19d (1.0 equiv) was reacted with Amberlyst-21 CuPF₆ (43.0 g,0.22 mmol/g, 1.0 equiv) in toluene (890 ml) for 16 h. Flashchromatography using silica (80% to 100% EtOAc in hexanes) provided thepure product (2S,5S,6S)-7d (8.01 g, 78%).

(2S,5S,6S)-7d: [α]_(D) ²⁰ +31.0 (c 5.9, CHCl₃).

Elaboration of Compounds to Final Core

Step 1:

2,6-Lutidine (2.5 mL, 21.4 mmol, 2.0 equiv) and TBSOTf (7.39 mL, 32.2mmol, 3.0 equiv) were added to a solution of 7a (16.0 g, 21.3 mmol, 1.0equiv) in CH₂Cl₂ (536 mL, 0.02 M) at 0° C. The mixture was warmed to RTand stirred for 2 h before being quenched with saturated NH₄Cl solutionand extracted with DCM. The combined organic extracts were dried overMgSO₄, filtered and concentrated to give 6.62 g of the crude silylcarbamate. The resulting oil was dissolved in DCM (107 mL) beforeHF.pyridine (70%, 2.23 mL, 10.7 mmol, 1.0 equiv) was added. The mixturewas stirred for 45 min, quenched with saturated NH₄Cl solution andextracted with EtOAc. The combined organic extracts were dried overMgSO₄, filtered and concentrated and taken directly to next step.

Step 2:

The crude material was dissolved in THF (186 mL) before 10% NaHCO₃solution (80 mL, excess) was added. The mixture was cooled to 0° C. andFmocCl (3.33 g, 12.86 mmol, 1.2 equiv was added, the mixture was stirredfor 30 min at 0° C. and then for an additional 4 h at RT. The reactionwas quenched with saturated aqueous NH₄Cl solution and the resultingmixture was extracted with EtOAc. The combined organic extracts weredried over MgSO₄, filtered and concentrated. Flash chromatography onsilica gel (80% to 100% EtOAc in hexanes) gave the product 28a (5.56 g,76% over 2 steps) as a white foamy solid.

Step 3:

(R,S,R)-28a (5.07 g, 7.44 mmol, 1.0 equiv) was dissolved in CH₂Cl₂ (119mL) and pH 7 buffer solution (30 mL). The mixture was cooled to 0° C.and dichlorodicyanobenzoquinone (3.38 g, 24.9 mmol, 1.5 equiv) wasadded. The mixture was stirred for 10 min at 0° C. and an additional 1 hat RT before being quenched with saturated NH₄Cl and diluted withCH₂Cl₂. The mixture was then filtered through celite, and the filtercake was washed several times with hot CH₂Cl₂. The combined organicextracts were washed with saturated NaHCO₃ solution before activatedcarbon was added. The filtrate was concentrated and flash chromatographyon silica gel (0% to 5% MeOH in CH₂Cl₂) gave pure product (2R,5S,6R)-31a(3.95 g, 97%).

(2R,5S,6R)-31: [α]_(D) ²⁰ +27.4 (c 8.6, CHCl₃). IR (cm⁻¹) 3382, 2929,1636, 1449, 759. ¹H NMR (500 MHz, DMSO-d₆, 130° C.) δ 7.95 (s, 1H), 7.84(d, J=7.1 Hz, 2H), 7.79 (d, J=7.0 Hz, 2H), 7.43-7.38 (m, 2H), 7.37-7.31(m, 2H), 6.20 (s, 2H), 4.69 (d, J=15 Hz, 1H), 4.56 (d, J=15 Hz, 1H),4.49-4.36 (m, 2H), 3.67-3.58 (m, 1H), 3.57-3.50 (s, 1H), 3.49-3.41 (s,1H), 3.30-3.32 (m, 1H), 2.80-2.72 (m, 1H), 2.63-2.54 (m, 1H), 2.41 (s,3H), 2.37-2.29 (m, 1H), 2.25-2.15 (s, 2H), 1.95-1.88 (m, 3H), 1.14 (d,J=6.3 Hz, 3H), 0.75 (d, J=6.4 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆, 130°C.) δ 171.5, 142.3, 138.9, 136.9, 128.0, 126.4, 124.9, 120.3, 119.0,107.8, 78.3, 63.3, 60.7, 53.6, 52.3, 45.9, 45.0, 48.5, 37.2, 35.6, 33.1,29.6, 24.3, 13.9, 10.9. HRMS (ESI) calcd for C₃₁H₃₅N₅O₅ [M+H]⁺:562.2951. Found: 562.3023.

Solid-Phase Library Synthesis:

General Methods:

Solid-phase synthesis was conducted on silicon-functionalizedpolystyrene SynPhase™ Lanterns (L-series) equipped with radio frequencytransponders (TranStems) for AccuTag directed sorting and compoundtracking. Quality-control Lanterns were included at each synthesis stepfor reaction monitoring by UPLC (UV 210 nM) after HF-cleavage. Allreactions were conducted in heavy wall pressure vessels from ChemGlasswith agitation in New Brunswick Scientific incubator shakers.

Scaffold Loading:

To a flame-dried flask containing silicon-functionalized Lanterns wasadded a freshly prepared solution of TfOH in anhydrous DCM (9.0 equiv, 5g of TfOH/100 mL of DCM) was added. Each flask was shaken at RT for 10min at which time the Lanterns had turned bright orange. The deep redTfOH solution was removed via cannula and anhydrous 2,6-lutidine (12.0equiv relative to Si) was added. Once the Lantern color had changed fromorange to white, the scaffold (1.2 equiv. relative to Si) was added as asolution in anhydrous DCM (0.4 mL/Lantern) and the reaction mixture wasshaken for 48 h overnight. The loading mixture was removed and set aside(to recover any unreacted alcohol) and the Lanterns were washed with thefollowing solvents for 30 min intervals: DCM, THF, 3:1 THF/IPA, 3:1THF/H₂O, DMF, 3:1 THF/H₂O, 3:1 THF/IPA, THF, DCM. The Lanterns were thendried on a lyophilizer overnight prior to sorting. All 8 stereoisomersof 29 were loaded via the same protocol.

Fmoc Removal:

To a flask containing Lanterns was added a solution of 20% piperidine inDMF (0.8 mL/Lantern). After shaking at RT for 30 min, the piperidinesolution was removed and the Lanterns were washed with the followingsolvents for 30 min intervals: DMF, 3:1 THF/H₂O, 3:1 THF/IPA, THF, DCM.The Lanterns were then dried on a lyophilizer overnight prior tosorting.

N-Capping/Acids:

To each flask containing lanterns was added DCM (0.8 mL/Lantern)followed by triethylamine (30 equiv) and the desired acid (20 equiv).PyBOP (20 equiv) was added and the Lanterns were shaken at RT overnightand then washed with following solvents for 30 min intervals: DCM, DMF,3:1 THF/H₂O, 3:1 THF/IPA, THF, DCM. The Lanterns were then dried on alyophilizer overnight prior to sorting

Cleavage Protocol:

To a 96-well plate containing Lanterns was added a 15% solution ofHF/pyridine in stabilized THF (350 μL/Lantern). After 2 h the cleavagesolution was quenched with TMSOMe (700 μL/Lantern) and the contents ofeach well were transferred to a pre-weighed 2-mL vial. The Lanterns werewashed with an additional 200 μL of stabilized THF (or THF/MeOH) and thesolution was transferred to the 2-mL vial. The samples were concentratedon a Genevac® solvent evaporation system overnight without heating.Loading masses for each alcohol was determined on a FlexiWeigh® system.

Compound 108 was synthesized following the general methods for librarysynthesis. Compound 31 was loaded onto Lanterns followed by subsequentFmoc removal. The secondary amine was capped with cyclopropanecarboxylic acid. Compound 108 was then cleaved from the Lanterns andisolated following the standard cleavage protocol.

Example 2: Design of a Primary Human Hepatocyte Platform for ChemicalScreening

In order to generate renewable sources of functional human hepatocytes,a high-throughput liver platform was developed to enable unbiasedchemical screening on primary human hepatocytes. The treatment of cellswith small molecules may modulate a wide range of cellular processes,such as stem cell self-renewal and differentiation, and proliferation ofnormally quiescent mature cells. Compounds can act through variousmechanisms to induce cell division, including activation ofdevelopmental signaling pathways such as Wnt or recruitment of GEFs tothe plasma membrane for RAS/MAPK pathway activation.

In order to avoid species-specific differences and cell line mutations,the screen was conducted with human primary hepatocytes. Traditionally,chemical screening on such cells has been hindered by their availabilityin large quantities as well as their rapid loss of viability andphenotype in vitro. Recent advances in cryopreservation technologiesallowed enough primary human cells to be stored for screening; thesecells are maintained through co-cultivation with non-parenchymal cells.Co-cultures of primary human hepatocytes with murine embryonic J2-3T3fibroblasts were recently shown to maintain normal hepatocyte phenotypefor weeks. These in vitro platforms consisted of hepatocytes surroundedby a co-planar population of fibroblasts. While sufficient forstabilizing hepatocyte functions in vitro, such platforms may limitnormal hepatocyte expansion due to contact inhibition. Thus, for thisscreen, a sparse population of hepatocytes was co-cultivated on top of aconfluent feeder layer of J2-3T3 fibroblasts within 384-well plates(FIG. 1A top). This screening platform stabilized hepatocyte phenotypicfunctions in vitro (FIG. 1A bottom) and was compatible with two separatehigh-throughput readouts developed for this screen.

The primary readout detected hepatocyte proliferation via automatedhigh-content imaging. This assay quantified hepatocyte nuclei numbers,using nuclear morphologies to separate the hepatocyte and fibroblastsubpopulations that co-exist within the screening platform. Whenvisualized with Hoechst stain, hepatocyte nuclei were smaller and moreuniform in texture while fibroblast nuclei were larger and punctated(FIG. 1A bottom). Leveraging this distinction, automated image analyseswas developed that utilized machine learning algorithms to classifynuclei types and tabulate hepatocyte nuclei numbers. Assay validationdata showed that this image-based readout can confidently (z′>0) detectdoublings in hepatocyte nuclei numbers with low variance (CV<20%) andgood reproducibility. Besides quantifying hepatocyte nuclei that havecompleted mitosis, the number of nuclei in the process of mitosis werealso quantified. Two additional analysis pipelines were built to detectnuclear morphologies consistent with cells undergoing metaphase andanaphase.

In order to evaluate the phenotype of treated cells, a secondary readoutwas included to quantify hepatocyte functions via competitive ELISA.This biochemical assay measured the level of secreted albumin as amarker for protein synthesis functions of the cultured hepatocytes (FIG.1A bottom).

Example 3: High-Throughput Identification of Small Molecules for HumanHepatocyte Sourcing

Using the high-throughput liver platform, 12,480 small moleculecompounds were screened. These molecules mainly comprised kinase-biasedcompounds (KinA) and commercial compounds (ComA), but also includebioactive compounds (BioA), chromatin-biased (CHRM) as well as a smallnumber of natural products (NatP). Of note in the library are ˜2,500diverse synthetic compounds unique to the Broad Institute. Thesecompounds rival the complexity of natural products, which is desirablebecause many small molecules known to affect biological processes arestructurally complex. Structural diversity is also desirable in thisscreen because every macromolecule in the cellular machinery is apotential target.

FIG. 1B top summarizes the workflow of the screen. Primary screening wasdone on human primary hepatocytes, preconditioned for seven days bynon-parenchymal cells in the screening platform. Upon stabilization invitro, cultures were treated with the chemical library for 48 hours. Allcompounds were tested in duplicates, at a single concentration of 15 μM.This single concentration was selected via a pilot screen of ˜2,000small molecules. All 12,480 small molecules were initially tested on asingle donor of cryopreserved hepatocytes (donor a) in order toeliminate noise from donor-to-donor variability. Following compoundtreatment, media supernatants were collected for functional analyses viaELISA and cultures were fixed in 4% paraformaldehyde for proliferationanalyses via imaging.

To identify proliferation hits, three image-based readouts—one each toquantify the number of (1) hepatocyte nuclei in interphase, (2) nucleiin metaphase and (3) nuclei in anaphase—were integrated. z Scores wereconverted into p values, which were then used to generate ranked listsof compounds based on the efficacy and consistency of effects across thedifferent proliferation readouts. Efficacy was assessed by the productof p values (p_(prod)=p_(inter)×p_(meta)×p_(ana)); consistency wasevaluated by the maximum of p values (p_(max)=max(p_(inter), p_(meta),p_(ana)). Compounds were considered proliferation hits ifp_(prod)<1×10⁻⁶ and p_(max)<0.25. Functional hits were selected by anELISA p<−0.05. Compounds with ELISA z>3.0 were eliminated as toxic. 93compounds met all hit selection criteria, qualifying as FunctionalProliferation Hits (FPH); FIG. 1B bottom illustrates the types ofcompounds that constituted this set of hits.

A total of 400 primary hits were retested in eight-point dose-responsecurves. These included 93 FPHs, as well as additional Proliferation-onlyHits (PHs, z>3.0 in any of the image-based readouts) and Function-onlyHits (FHs, z<−4.2 in ELISA). A different donor of cryopreservedhepatocytes (donor B) was used during retest in order to includebiological diversity in the screen. Remaining hits were filtered througha cell-free counter screen to eliminate compounds that interfered withthe ELISA assay chemically. Ultimately, twelve confirmed hits wereobtained.

Among these confirmed hits were two classes of compounds, which enabledtwo different approaches for generating a renewable source of functionalhuman hepatocytes. One class of hits (FPHs) induces functionalproliferation of hepatocytes in vitro, and can thus be used to expandmature human primary hepatocytes. The second class of hits (FHs)enhances the functions of cultured hepatocytes, and thus can be used todifferentiate iPS-derived hepatocytes toward a more differentiatedphenotype.

Example 4: Expansion of Human Primary Hepatocytes

The ability of compounds of the invention (FPHs) to expand human primaryhepatocytes in vitro was tested. Prostaglandin E2 (PGE2), which promotesliver regeneration in zebrafish via Wnt signaling, was used as apositive control. PGE2 was tested on human primary hepatocytes in thehigh-throughput liver platform described herein and found it to be aFPH. Two other compounds of the invention (strong FPHs (FPH1 and FPH2)),identified through unbiased screening, were tested. Both compoundsinduced a 1.5× increase in hepatocyte nuclei numbers during primaryscreening (FIG. 2A), elevated the number of nuclei undergoing mitosis(FIG. 2B), and these effects on hepatocytes were dose-responsive (FIG.2C). Cells treated with these compounds also maintained theirliver-specific functions.

To characterize the effects of FPH1 and FPH2 outside of the screeningplatform, human primary hepatocytes were seeded at a density of ˜20,000cells/cm², into standard 12-well tissue culture plates on top of afeeder layer of growth-arrested J2-3T3s. These cells were cultured for 7days, during which time a single compound/FPH was supplemented into themedia on days 1 and 5 at a concentration of 20 μM. Treated hepatocytecolonies increased in size over time, with more hepatocytes populatingeach colony (FIG. 2D, 2E). Furthermore, compound/FPH treatment increasedKi67 staining, which not only co-localized with Hoechst stains for cellnuclei but also with human albumin stains for hepatocytes (FIG. 2E).Quantitative image analysis showed an up to 6.6- and 3.5 fold increasesin the area of albumin-positive colonies upon FPH1 and FPH2 treatment,respectively (FIG. 2F). The vast majority of Ki67-positive nucleiexhibited hepatocyte nuclear morphologies, which was consistent with thelack of proliferating cells in fibroblast-only cultures treated withFPHs. These results strongly indicated that human primary hepatocytescan be induced to proliferate in vitro using FPHs.

To characterize the degree and kinetics of proliferation, the number ofhepatocytes in culture was quantified using both an automated cellcounter and FACS analysis. Results showed a dramatic, up to 10-foldincrease in the number of hepatocytes when treated with various FPHs(FIG. 2G). This difference in effect, however, may not reflect compoundefficacy; the number of Ki67-positive nuclei was more elevated with FPH2treatment, suggesting that FPH2 effects were simply delayed. The vastmajority of Ki67-positive nuclei exhibited hepatocyte nuclearmorphologies, which is consistent with the lack of proliferating cellsin fibroblast-only cultures treated with FPHs. These results stronglyindicated that human primary hepatocytes can be induced to proliferatein vitro using FPHs.

To characterize the degree of proliferation and to obtain a doublingtime, cells were counted and fluorescence-activated cell sorting (FACS)was performed (FIG. 2H). For cell counting, FPH-treated cells weretrypsinized after seven days in culture. The number of cells wasquantified using an automated cell counter. The number of hepatocyteswas obtained by subtracting the number of fibroblasts from the totalnumber of cells found in the corresponding co-culture. For FACSanalysis, growth arrested J2-3T3s were labeled with CM-DiI prior toinitiation of co-culture so that hepatocytes can be identified vianegative selection. To enable cell counting during FACS, each sample wassupplemented with fluorescent counting beads. The strongestproliferation inducer was FPH1 (FIG. 2G bottom graph). Over 7 days, FPH1induced hepatocyte doublings at a rate that is consistent with reportedliver regeneration kinetics in vivo.

To assess the phenotype of the treated hepatocytes, imaging, biochemicalanalyses and gene expression profiling were performed. Phase contrastimaging was used to monitor hepatocyte morphology and found that normalmorphology was maintained throughout the treatment period (FIG. 2D).Albumin secretion and urea synthesis, a surrogate marker of nitrogenmetabolism, were both stable throughout FPH treatment (FIG. 3A top andmiddle). Metabolic functions were assessed via examinations ofcytochrome P450 (CYP450) activity and canalicular transport. Resultsshowed normal CYP450 activities (FIG. 3A bottom) and active transport ofa fluorometric substrate into the bile cannaliculi between hepatocytes(FIG. 3B). Gene expression profiling confirmed that there are nosignificant differences between FPH-treated and untreated hepatocytes(FIG. 3C). These results agree with literature findings of sustainedliver functions throughout liver regeneration.

Example 5: Differentiation of Human iPS-Derived Hepatocytes

The ability of compounds of the invention to affect the differentiationand maturation of iHeps was tested. Undifferentiated iPS cells werecultured on Matrigel, supported by primary mouse embryonic fibroblasts.Once confluent, iPS cells were transitioned to differentiation media,with sequential addition of growth factors (Activin A, BMP-4, bFGF, HGF,and OSM) to guide differentiation, first into endoderm, then intohepatic progenitor cells and finally into iHeps. Compound treatmentstarted on day 21 post initiation of differentiation and acted over aperiod of 9 days. FH1 and FPH2 were used to treat iHeps. FH1 doubledalbumin secretion during primary screening (FIG. 4A left) in adose-responsive manner (FIG. 4A right). FPH2 had similar, but weakereffects on hepatocyte functions.

iHeps treated with FH1 and FPH2 developed more mature hepatocytephenotypes. Colonies of hepatocyte-like cells increased in size withcompound treatment (FIG. 4B), suggesting more differentiation of iPScells down a hepatic lineage. Treated colonies also exhibited morepronounced hepatocyte morphologies, with more noticeable bilecannaliculi between hepatocytes indicated by yellow arrows in FIG. 4B.

Gene expression profiles showed that treated iHeps more closely resemblemature hepatocytes than untreated cells (FIG. 4C). Euclidian clusteringanalyses groups untreated cells with fetal hepatocytes and treated cellswith mature hepatocytes (FIG. 4C left). Of particular interest are ABCtransporters, CYP3A4 and GSTP1 expression levels. ABC transporters areknown to mature after birth. ABCB11, also known as bile-salt export pump(BSEP), increased ˜3 fold and ˜4 fold in expression levels with FPH2 andFH1 treatment, respectively, compared to untreated iHeps. In contrast,GSTP1 expression, whose levels decrease with maturity, remained low uponFPH2 and FH1 treatment compared to untreated iHeps (FIG. 4C right).

To examine the effects of FH1 and FPH2 at the protein level, AFP,albumin and CYP3A4 levels weew visualized via immunofluorescentstaining. Images showed dramatic increases in albumin staining upon bothFH1 and FPH2 treatment, although the effects of FH1 were more pronounced(FIG. 4D top). This is in agreement with primary screening results aswell as earlier morphological findings. Untreated cultures had islandsthat double stain for albumin and the fetal marker AFP, with very littlepresence of the mature marker CYP3A4. In contrast, FH1-treated islandsdouble stained strongly for albumin and CYP3A4, with very minimalpresence of AFP.

To confirm the staining results, secreted levels of albumin and AFP viaELISA, and CYP450 activities through isoenzyme-specific probes that areeither fluorescent or luminescent were measured. ELISA results verifiedboth an increase in albumin secretion and a decrease in AFP secretion bytreated iHeps (FIG. 4D bottom). CYP3A4 activity increased by 16 and 45times upon treatment with FH1 and FPH2, respectively. CYP2A6, anothermature CYP450, also increased significantly upon treatment with compound(FIG. 4E). Induction was considered as a possible explanation for theseelevations in CYP450 activity. However, this was unlikely based on thefollowing experiments. While human hepatocytes treated with 250 μMβ-naphthoflavone (BNF) exhibited elevated CYP450 activity (FIG. 4Fleft), such elevations were mostly lost 24 hrs after removal of theinducer (FIG. 4F right). Since a period of at least 48 hrs separatedcompound treatment and the measurement of CYP450 activity, the iHepswere expected to have recovered from any general elevations in CYP450activity.

The more mature phenotype exhibited by treat iHep cells was stable forat least 1 week after removal of FH1 and FPH2. iHeps were treated withFH1 and FPH2 once on day 20 and then maintained in normal basal media(without FH1 and FPH2 addition) for 9 days. Phase contrast andimmunofluorescent stained images for Albumin, CYP3A and AFP showed thatcells maintained a mature phenotype at Day 29 in culture (FIG. 5A).Quantification of secreted albumin, AFP, and CYP3A4 and CYP2A6 activityfurther demonstrated the stability of mature iHep phenotype (FIGS.5B-5C).

Together, these results showed that FH1 and FPH2 were able to matureiHeps beyond what is currently achievable using defined factors only,thus alleviating a major obstacle to the use of iPS cells as a source offunctional human hepatocytes.

Example 6: Expansion of Multiple Different Donors of Primary HumanHepatocytes

To generalize the findings of compounds of the invention across multipledonors, primary human hepatocytes from six additional cell sources weretreated with FPH1 and FPH2 and stained for Ki67 and albumin after sixdays in culture Immunofluorescent staining showed that treatment withFPH1 and FPH2 resulted in increased Ki67 staining that co-localized withalbumin labeled hepatocytes (FIG. 6A). On day seven, the number ofhepatocytes was quantified by FACS analysis (FIG. 6B). Day one untreatedcontrols was added to the bar graph for reference. Both compoundsinduced an increase in the number of hepatocytes in all donor celltreated. These results together suggest that the FPHs are active acrossa wide range of genetically diverse individuals.

Example 7: Generation of iHeps

iHep cells were generated from iPS cells using the methodology describedherein (FIG. 7A) Immunostaining of iPS and iHep cells with hepaticlineage marker showed that iHeps express alpha-1-Antitrypsin andCytokeratin 18, whereas undifferentiated iPS cells do not (FIG. 7B).FACS analysis profiling hepatic progenitor markers (EPCAM and DLK) andimmature markers (SSEA1 and Oct 4) illustrated that iHeps exhibithepatic-like progenitor cell profile in contrast to the undifferentiatedprofile exhibited of iPS cells (FIG. 7C).

Example 8: Augmentation of Liver Development

The effects of FH1 and PH1 on liver development were examined intransgenic lfabp:GFP zebrafish, in which GFP expression is restricted tothe liver. Zebrafish embryos were allowed to develop normally for 24 hrspost fertilization and then exposed to either FH1 or PH1 until 72 hrspost fertilization (FIG. 8A). Livers were visualized by whole-mountfluorescence imaging (FIG. 8B) and lfabp in situ hybridization (FIG.8C). Qualitative assessed of liver formation by fluorescent microscopyindicated that treatment with FH1 and PH1 increased liver size comparedto DMSO-treated controls (FIG. 8D).

Example 9: Protection Against Acute Liver Injury

The effect of FH1 on zebrafish survival following APAP-induced toxicitywas examined. lfabp:GFP zebrafish embryos were allowed to developnormally for 72 hrs post fertilization and then exposed to a fatal doseof 10 mM APAP concurrently with FH1 (FIG. 9A). FH1 treatment mitigatedAPAP-induced death resulting in the survival of 71% of FH1-treatedembryos compared to 16% of controls (FIG. 9B). Similar protectiveeffects of FH1 were seen in adult lfabp:GFP zebrafish (FIGS. 9C-9D).

The current clinical treatment for APAP toxicity is the administrationof NAC, which has a limited window of efficacy. To examine thetherapeutic window of FH1 and PH1 efficacy, lfabp:GFP zebrafish embryoswere allowed to develop normally for 48 hrs post fertilization, and thenexposed to a non-fatal dose of 5 mM APAP for 24 hrs before initiation oftreatment with FH1 or PH1 for an additional 24 hour period (FIG. 10A).At 96 hrs post-implantation, whole-mount fluorescence imaging showedthat both FH1 and PH1 mitigated APAP-induced loss of liver massresulting in enhanced embryonic liver size compared to controls (FIG.10B). Moreover, efficacy of both FH1 and PH1 exhibited an elongatedtherapeutic window of least 24 hrs, compared to the typical ˜8 hrtherapeutic window for NCA.

Example 10: Expansion of Compound-Treated Human Primary Hepatocytes

The ability of compounds 106-108 to expand human primary hepatocytes invitro was tested. Cell counts of treated cultures showed that the numberof hepatocytes increased in number relative to controls (FIG. 11A). Themetabolic phenotype of the treated hepatocytes was assessed forcanalicular transport. Results showed normal active transport of afluorometric substrate into the bile cannaliculi between hepatocytes(FIG. 11B) and CYP450 activity (FIG. 11C), indicating that cellsexhibited normal liver-specific functions. Gene expression profilingconfirmed that there was no significant differences between FPH-treatedand untreated hepatocytes (FIG. 11D). Furthermore, compound 107treatment increased Ki67 staining, which not only co-localized withHoechst stains for cell nuclei but also with human albumin stains forhepatocytes (FIG. 11E), further indicating that compound 107 inducedproliferation of human primary hepatocytes.

The ability of compounds 201-212, 215, 217 and 244-258 to expand humanprimary hepatocytes in vitro was also tested. Cell counts of treatedcultures showed that the number of hepatocytes increased in numberrelative to controls (FIG. 11F).

Example 11: Differentiation of Human iPS-Derived Hepatocytes

The ability of compounds 106 and 108 to affect the differentiation andmaturation of iHeps was tested. Undifferentiated iPS cells were culturedon Matrigel, supported by primary mouse embryonic fibroblasts. Onceconfluent, iPS cells were transitioned to differentiation media, withsequential addition of growth factors (Activin A, BMP-4, bFGF, HGF, andOSM) to guide differentiation, first into endoderm, then into hepaticprogenitor cells and finally into iHeps. Compound treatment started onday 21 post initiation of differentiation and acted over a period of 9days.

To determine the effects of compounds 106 and 108, secreted levels ofalbumin and AFP were measured by ELISA. Results showed that treatediHeps exhibited increased levels of albumin secretion and decreasedlevels of AFP secretion (FIG. 12A). To examine the effects of compound106 at the protein level, albumin and AFP were visualized viaimmunofluorescent staining. Images showed dramatic increases in albuminstaining upon treatment with compound 106 (FIG. 12B). Consistent withthe literature, untreated cultures had islands that double stain foralbumin and the fetal marker AFP, with very little presence of themature marker Albumin. These results demonstrated that iHeps treatedwith compounds 106 and 108 develop more mature hepatocyte phenotypes.

The ability of compounds 201-212, 217 and 244-258 to affect thedifferentiation of iHeps was also tested. As evidenced by the increasein CYP3A4 activity in treated iHeps relative to controls (FIG. 12C),these compounds induced differentiation of iHeps.

Example 12: Differentiation of iPS-Derived Endothelial Cells

In analogy with the iPS-derived hepatocytes of Example 5, iPS-derivedendothelial cells were treated with compounds of the invention to assesstheir ability to induce differentiation. Cells were assayed for nitricoxide (NO), a mature marker for endothelial cells. CB=FPH2, M=FH1,E=PH1, while Null and DMSO are control runs with no additive and vehicleonly, respectively. Results are depicted in FIG. 13, showing that FPH2,FH1, and PH1 all induce differentiation of these cells.

Example 13: Functional Analysis of Human Primary Hepatocytes Treatedwith Compound 102

Compounds of the invention were screened for their ability to increasehepatocyte nuclei count. Compound 102 induced a significant increase inhepatocyte nuclei count (proliferation Z score of 2.24), thus qualifyingit as a proliferation-only hit. FPH2 and Compound 102 both share thesame 5-chloro-2-methyl substitution on the sulfonamido phenyl ring.

Example 14: Kinome Analysis

Selected compounds of the invention (FPH1 and FPH2; both at 1 μMconcentration) were screened against a panel of kinases (FIG. 17).

FPH1 inhibited the activity of the following kinases by 18.4-30.0%(i.e., 70.0-81.6% activity remaining): FMS (23% inhibition; 77%remaining activity) and IGF-1R (21% inhibition; 79% remaining activity).FPH1 inhibited the activity of the following kinases by 6.7-18.4% (i.e.,81.6-93.3% activity remaining): MAPKAP-KII, MAPKAPKV (PRAK), Pim1, CK1γ2, GRK1, PAK5, MSSK1, SAPK4, eEF-2K, EphB4, KDR and PI3KCγ.

FPH2 inhibited the activity of the following kinases by 18.4-30.0%(i.e., 70.0-81.6% activity remaining): FMS (28% inhibition; 72%remaining activity), ALK (23% inhibition; 77% remaining activity) andEphB4. FPH2 inhibited the activity of the following kinases by 6.7-18.4%(i.e., 81.6-93.3% activity remaining): CaMK1 δ, RSK2, MAPKAPKV (PRAK),RSK3, RSK4, CK1 γ2, PI3KCγ, PI3KC2 α, PDGFRβ, Ron, FAK, eEF-2K, CLK1,CLK4, NEK2, NEK9, CDK5 and DAPK1.

FMS is a member of the CSF1R/PDGF receptor family of tyrosine kinases,and mediates biological effects of CSF1, which affects production,differentiation and cell function of monocyte lineage. Mutations in theFMS kinase are associated with myeloid malignancy.

Example 15: Physico-Chemical Characterization

Selected compounds of the invention were evaluated for physico-chemicalproperties, including solubility, microsomal stability, protein bindingand plasma stability (Table 2).

TABLE 2 Physico-chemical properties. Solubility (μM) - PBS with 1% DMSOCompound Rep1 Rep2 Rep3 Average BRD-K05085281 23 28 24 25BRD-K17976466 >100 >100 >100 >100 BRD-K37628956 33 47 41 41 ControlsAntipyrine >100 >100 >100 >100 Clotrimazole 1.6 1.9 3.1 2.2 Microsomalstability (% remaining) - Human Compound Rep1 Rep2 Average No NADPHBRD-K05085281 53.9 58.1 56.0 104.2 BRD-K17976466 99.4 95.3 97.4 102.3BRD-K37628956 52.4 59.3 55.8 94.4 Controls Atenolol 103.1 97.8 100.492.1 Verapamil 6.6 7.1 6.9 92.8 Protein binding (% bound) - HumanCompound Rep1 Rep2 Average BRD-K05085281 99.3 99.3 99.3 BRD-K1797646611.8 12.1 12.0 BRD-K37628956 99.4 99.5 99.5 Controls Verapamil 92.0 92.992.4 Lidocaine 58.2 60.7 59.5 Plasma stability (% remaining) - HumanCompound Average BRD-K05085281  98.0 BRD-K17976466 104.2 BRD-K37628956 87.1 Controls Verapamil  98.5 Eucatropine  0.5 Note: average percentremaining represents disappearance through both enzymatic andnon-enzymatic microsomal mechanisms. “No NADPH” result representsdisappearance through just the non-enzymatic microsomal mechanisms.Batch of BRD-K05085281 used: BRD-K05085281-001-03-5 Batch ofBRD-K17976466 used: BRD-K17976466-001-03-8 Batch of BRD-K37628956 used:BRD-K37628956-001-03-8

Example 16: Physico-Chemical Characterization

Selected compounds of the invention were evaluated for physico-chemicalproperties, including solubility, microsomal stability, protein bindingand plasma stability (Table 3).

TABLE 3 Physico-chemical properties. Solubility (μM) - PBS with 1% DMSOCompound Rep1 Rep2 Rep3 Average BRD-K61250484-001-01-5 23 22 26 23BRD-K94248251 23 16 21 20 BRD-K44777625-001-01-9 45 39 36 40BRD-A07207424-001-04-1 112 114 104 >100 Controls Antipyrine 89 105 86 93Clotrimazole 3.8 2.1 2.0 2.6 Microsomal Stability (% remaining) - HumanCompound Rep1 Rep2 Average No NADPH BRD-K61250484-001-01-5 4.2 4.5 4.4110.6 BRD-K94248251 41.7 39.4 40.6 123.1 BRD-K44777625-001-01-9 71 66.768.9 63 BRD-A07207424-001-04-1 81.5 85.5 83.5 100.3 Controls Atenolol107.3 104.1 105.7 105.5 Verapamil 5.0 4.9 5.0 111.3 Protein Binding (%bound) - Human Compound Rep1 Rep2 Average BRD-K61250484-001-01-5 90.991.6 91.2 BRD-K94248251 95.7 95.9 95.8 BRD-K44777625-001-01-9 91.0 91.091.0 BRD-A07207424-001-04-1 76.2 75.0 75.6 Controls Verapamil 96.3 96.296.3 Lidocaine 75.7 76.0 75.8 Plasma Stability (% remaining) - HumanCompound Average BRD-K61250484-001-01-5 101.4 BRD-K94248251  63.9BRD-K44777625-001-01-9 100.6 BRD-A07207424-001-04-1 105.9 ControlsVerapamil 108.4 Eucatropine  0.4 Note: The average percent remainingrepresents disappearance through both enzymatic and non-enzymaticmicrosomal mechanisms. The “No NADPH” result represents disappearancethrough just the non-enzymatic microsomal michanisms. Batch ofBRD-K61250484 used: BRD-K61250484-001-01-5 Batch of BRD-K44777625 used:BRD-K44777625-001-01-9 Batch of BRD-A07207424 used:BRD-A07207424-001-04-1

Example 17: In Vivo Therapeutic Effects

The In Vivo Therapeutic Effects of Selected Compounds of the Inventionwere evaluated in a zebrafish model of acetaminophen overdose. Zebrafishembryos were co-administered a lethal dose of acetaminophen and acompound of the invention. The therapeutic benefit of the compound ofthe invention was assessed through survival of the embryos at 24 hourspost exposure to the compounds. Results are illustrated in FIG. 18.

Example 18: In Vitro Maturation Effects

The in vitro maturation effects of selected compounds of the inventionon human iPS-derived hepatocyte-like cells (iHeps) were evaluated. HumaniPS cells were differentiated into iHeps and treated with selectedcompounds of the invention for 9 days. Maturity of treated iHeps wasevaluated through ELISA quantification of mature (albumin) and immature(AFP) markers. Results are illustrated in FIG. 19.

The results illustrated in FIG. 19A (albumin) were obtained with the DOS1 concentration of 6.7 μM, and the DOS 3 concentration of 10 μM. Theexperimental range of effective concentrations was 5-13.3 μM for DOS 1,and 5-20 μM for DOS 3.

The results illustrated in FIG. 19B (AFP) were obtained with the DOS 1concentration of 10 μM, and the DOS 3 concentration of 20 μM. Theexperimental range of effective concentrations was 10-40 μM for DOS 1and 10-40 μM for DOS 3.

Example 19: Cytochrome P450 Activity

iPS Cells were differentiated into iHeps, treated with selectedcompounds of the invention for 9 days, then assayed for CYP functions.The general CYP activity of the iHeps treated with compounds of theinvention were elevated (FIG. 20A). The treated iHeps displayed elevatedmature CYP activity, both for CYP3A4 (FIG. 20B) and CYP2A6 (FIG. 20C).On the other hand, the treated iHeps displayed depressed immature CYPactivity, as demonstrated for CYP3A7 (FIG. 20D).

The results illustrated in FIG. 20A (general BFC) were obtained with theDOS 1 concentration of 10 μM, and the DOS 3 concentration of 13.3 μM.The experimental range of effective concentrations was 6.7-13.3 μM forDOS 1, and 13.3 μM for DOS 3.

The results illustrated in FIG. 20B (CYP3A4) were obtained with the DOS1 concentration of 10 μM, and the DOS 3 concentration of 10 μM. Theexperimental range of effective concentrations was 6.7-13.3 μM for DOS1, and 6.7-20 μM for DOS 3.

The results illustrated in FIG. 20C (CYP2A6) were obtained with the DOS1 concentration of 13.3 μM, and the DOS 3 concentration of 26.7 μM. Theexperimental range of effective concentrations was 10-20 μM for DOS 1,and 10-26.7 μM for DOS 3.

The results illustrated in FIG. 20D (CYP3A7) were obtained with the DOS1 concentration of 10 μM, and the DOS 3 concentration of 10 μM. Theexperimental range of effective concentrations was 6.7-10 μM for DOS 1,and 10-13.3 μM for DOS 3.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method of inducing differentiation of one ormore induced pluripotent stem cell (iPS cell)-derived, immaturehepatocyte cells (iHep cells), the method comprising: contacting the oneor more iHep cells with compound 105(N,N′-(methylenebis(4,1-phenylene))diacetamide); or a pharmaceuticallyacceptable salt thereof; wherein said contacting with the compoundinduces differentiation of the one or more immature iHep cells intomature hepatocytes.
 2. The method of claim 1, wherein the maturehepatocytes derived from the one or more iHep cells contacted withcompound 105 express elevated levels of albumin in culture.
 3. Themethod of claim 1, wherein the mature hepatocytes derived from the oneor more iHep cells contacted with compound 105 express minimal levels ofalpha fetoprotein (AFP) in culture.
 4. The method of claim 1, whereinthe mature hepatocytes derived from the one or more iHep cells contactedwith compound 105 express elevated levels of CYP34A and CYP2A6 activityin culture.
 5. The method of claim 1, wherein the one or more iHep cellsare human iHep cells.
 6. The method of claim 1, wherein the one or moreiHep cells is contacted with compound 105 ex vivo.
 7. A method ofinducing differentiation of one or more induced pluripotent stem cell(iPS cell)-derived endothelial cells, the method comprising contactingthe one or more iPS cell-derived endothelial cells with compound 105(N,N′-(methylenebis(4,1-phenylene)) diacetamide), or a pharmaceuticallyacceptable salt thereof, wherein said contacting with the compoundinduces differentiation of the one or more iPS cell-derived endothelialcells to produce a population of more mature nitric oxide-producingendothelial cells.
 8. The method of claim 7, wherein the one or more iPScell-derived endothelial cells is contacted with compound 105 ex vivo.